Methods and assays for extraction-free detection of nucleic acids in a biological sample

Abstract

This application generally relates to methods and assays for detecting the presence or absence of a target nucleic acid in a biological sample, which in some embodiments are "extraction-free" methods.

Description

METHODS AND ASSAYS FOR EXTRACTION-FREE DETECTION OF NUCLEIC ACIDS IN A BIOLOGICAL SAMPLE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U. S. C. §119(e) to U. S. Provisional Application No. 63 / 742,802. filed January 7, 2025. The contents of the prior application are considered part of and are hereby incorporated by reference in their entirety.BACKGROUND

[0002] The emergence of circulating cell-free DNA (ccfDNA) (e g., fetal DNA in maternal plasma (ccffDNA). or cell-free tumor DNA) has transformed nucleic acid detection, which is important for both medical and non-medical purposes (e.g., fetal sex determination, cancer diagnostics, etc.). However, conventional methods present significant challenges. They require greater blood volumes, leading to inconvenience in patient draw and storage, as well as generating substantial biohazard waste post-processing. Moreover, these methods are prone to genomic DNA (gDNA) carryover from the buffy coat, entail a time-consuming DNA isolation process, and pose risks of contamination or inconsistency. Additionally, the presence of polymerase chain reaction (PCR) inhibitors in purification ingredients further complicates the procedure, prolonging processing time and reducing DNA yield and integrity. Furthermore, comparability issues arise due to variations in cfDNA values across different studies, while the necessity for expensive instrumentation adds to the complexity and cost. Improved methods of nucleic acid detection are needed.SUMMARY

[0003] Novel methods and assays are described herein which address one or more of these challenges. In some embodiments, the disclosed methods offer a more efficient and reliable approach to fetal sex determination. In some embodiments, the disclosed methods and assays are applicable to pathological conditions, such as cancer. In some embodiments, particularly where rare target genes are present, eliminating purification steps that cause significant loss of input material can be important and distinctive when it comes to early disease detection. The disclosed methods and assays include the following numbered embodiments.1. A method of detecting the presence or absence of a target nucleic acid in a biological sample, the method comprising:a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample;-1- ACTIVE\1627041887.1b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume); and c) performing a quantitative polymerase chain reaction (qPCR) using all or a portion of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample.2. The method of embodiment 1, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40.3. The method of embodiment 1 or 2, wherein the treatment is a proteinase, optionally proteinase K.4. The method of any one of embodiments 1-3, wherein terminating the treatment is inactivation of the treatment, optionally inactivation of the proteinase.5. The method of embodiment 4, wherein inactivation of the treatment comprises incubation of the biological sample at an inactivation temperature for a first period of time.6. The method of embodiment 5, wherein the inactivation temperature is less than about 65 °C.7. The method of embodiment 5 or 6, wherein the inactivation temperature is about 55 °C or about 45-65 °C, 45-60 °C, 50-65 °C, 50-60 °C, or 52-57 °C.8. The method of any one of embodiments 5-7, wherein the first period of time is about 10 minutes or about 5-25 minutes, 5-20 minutes, 10-25 minutes, 5-15 minutes, or 8-12 minutes.9. The method of any one of embodiments 1-8, wherein the method further comprises activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a second period of time.10. The method of embodiment 9, wherein the activation temperature is greater than 20 °C.11. The method of embodiment 9 or 10, wherein the activation temperature is about 37 °C, or about 20-42 °C, 27-42 °C, 27-40 °C, 30-42 °C, 30-40 °C, or 35-40 °C.12. The method of any one of embodiments 9-11, wherein the second period of time is about 30 minutes, or about 10-50 minutes, 10-40 minutes, 20-50 minutes, 20-40 minutes, or 25-35 minutes.13. The method of any one of embodiments 1-12, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment in step a).14. The method of any one of embodiments 1-13, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b).-2- ACTIVE\1627041887.115. The method of any one of embodiments 1-14, wherein upon completion of step b) or wherein the biological sample on which the qPCR is performed, the biological sample is diluted to about 1:1.25, 1:1.5, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, or a range defined by any two of the preceding values, optionally 1:1.25 to 1:35, 1:2 to 1:20, or 1:5 to 1:10.16. The method of any one of embodiments 1-15, wherein the biological sample is diluted to about 1:10.17. The method of any one of embodiments 1-16, wherein:wherein said applying a treatment to a biological sample comprises treatment with proteinase K and incubating the biological sample at about 37 °C for about 30 minutes;wherein said terminating the treatment to the biological sample comprises incubating the biological sample at about 55°C for about 10 minutes; and wherein upon completion of step b) the biological sample is diluted to about 1:10.18. The method of any one of embodiments 1-17, wherein step a) further comprises applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is sodium dodecyl sulfate (SDS), optionally wherein the detergent is applied to the biological sample at a concentration that is, is about, is not more than, is not less than, 0.001, 0.002, 0.005, 0.01, 0.02. 0.05, 0.1, 0.2, 0.5, 1. 2, 5, 10, 12, 15, or 20%, or a range defined by any two of the preceding values, for example. 0.001-20, 0.001-1. 0.002-2, 0.05-10. 0.1-10, or 0.002-0.5%, optionally 0.005-0.2%, or 0.02%.19. The method of any one of embodiments 1-18, wherein dual-labeled probes are utilized in the qPCR of step c, optionally wherein the dual-labeled probes each comprise a minor groove-binding moiety.20. The method of any one of embodiments 1-19, wherein the biological sample volume is less than about 500 pL, optionally wherein the biological sample volume is less than about 100 pL.21. The method of any one of embodiments 1-20. w-herein the biological sample volume is about 10 pL or about 1-500 pL, 2-500 pL, 5-500 pL. 10-500 pL. 100-400 pL, 1-100 pL, 5-50 pL, 50-100 pL, 5-20 pL, 5-15 pL, or about 1-12 pL.22. The method of any one of embodiments 1-21, wherein the biological sample is blood, plasma, serum, saliva, urine, and / or cervical mucus, optionally wherein the biological sample is treated with a preservative.-3- ACTIVE\1627041887.123. The method of any one of embodiments 1-21, wherein the biological sample is plasma.24. The method of embodiment 23, further comprising separating the plasma from a whole blood sample using centrifugation, optionally wherein the whole blood sample comprises capillary blood.25. The method of embodiment 24, wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL, optionally wherein the plasma sample volume is about 2 pL, and the whole blood sample volume is about 5 pL.26. The method of embodiment 24, wherein the plasma sample volume comprises about 1-100 pL, and the blood sample volume comprises about 2.5-500 pL.27. The method of any one of embodiments 1-26, wherein the target nucleic acid is DNA or RNA.28. The method of any one of embodiments 1-27, wherein biological sample is from a pregnant subject and the target nucleic acid is a fetal nucleic acid.29. The method of any one of embodiments 1-27, wherein the sample is from a subject, and the target nucleic acid is a genomic nucleic acid of the subject, optionally wherein the target nucleic acid is a tumor cell-derived nucleic acid.30. The method of any one of embodiments 1-29, wherein the target nucleic acid is a cell-free nucleic acid, optionally wherein the target cell-free nucleic acid is a cell-free DNA (cfDNA).31. The method of embodiment 30, wherein the biological sample comprises a concentration of total cfDNA per sample volume (ng / mL), optionally wherein the concentration of total cfDNA per sample volume is 0.0003, 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any two of the preceding values, for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, 0.01-3 ng / mL, or 0.03-2 ng / mL, optionally 0.5 ng / mL.32. The method of embodiment 31, wherein the target nucleic acid comprises 2-10% of the total abundance / amount of cfDNA in the biological sample.33. The method of any one of embodiments 1-32, wherein the biological sample comprises a concentration of target nucleic acid per sample volume (ng / mL), optionally wherein the concentration of target nucleic acid per sample volume is 0.0003, 0.0006, 0.001, 0.003, 0.006. 0.01, 0.03, 0.06, 0.1. 0.2, 0.3, 0.4. 0.5. 0.6, 0.7, 0.8, 0.9. 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any two of the -4- ACTIVE\1627041887.1preceding values, for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, 0.01-3 ng / mL, or 0.03-2 ng / mL. optionally 0.5 ng / mL.34. The method of any one of embodiments 1-33, wherein the biological sample comprises 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, or 15000 copies of the target nucleic acid per sample volume (copies / pL) or a range defined by any two of the preceding values, for example 0.1-15000, 1-5000, 1-1000, 5-10000, 5000-15000, 10-5000. 1-200. or 1-50 copies / pL. optionally 15-3000 copies / pL or about 300 copies / pL.35. The method of any one of the preceding embodiments, wherein the concentration of the target nucleic acid in the biological sample is, or is less than, 3000, 2500, 2000, 1500, 1000, 500, 250, 100, 50, 25, 10, 9, 8. 7, 6, 5, 4. 3, 2, 1. 0.5, or 0.1 GE / mL of biological sample, or a range defined by any two of the preceding values, for example 0.1-3000, 500-2500, 1-3000, 0.1-500, 0.1-100, 0.1-50, 1-10, 1-5, 0.5-10, 0.5-5 GE / ml ofbiological sample, optionally 10-500 GE of target nucleic acid per rnL of sample.36. The method of any one of embodiments 1-35, wherein the target nucleic acid is associated with a phenotypic trait.37. The method of any one of embodiments 1-36, wherein the target nucleic acid is on the Y-chromosome, optionally wherein the the target nucleic acid is present as, as about, as not less than, or as not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500. 1000, 2000, 3000. 4000, 5000, 6000, 7000. 8000, 9000, or 10000 copies on the Y-chromosome, or a range defined by any two of the preceding values, for example 1-10000, 1-6000, 4000-10000, 2000-8000, 1-500, 1-100, 1-10, 10-100, or 1-50 copies per Y-chromosome, optionally 2000-8000, or 4500-6000 copies per Y-chromosome.38. The method of any one of embodiments 1-37, wherein the target nucleic acid is associated with a pathological condition, optionally wherein the pathological condition is a congenital disease or cancer.39. A method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of embodiments 1-37, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, optionally wherein the whole blood sample is a capillary sample, wherein the target nucleic acid is a fetal cell-free nucleic acid, andwherein the target nucleic acid is on the Y chromosome.-5- ACTIVE\1627041887.140. The method of embodiment 39, the method further comprising determining the sex of the fetus, wherein:i) the presence of the target nucleic acid is detected and the fetus is determined to be male, orii) the absence of a target nucleic acid is detected and the fetus is determined to be female.41. A method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of embodiments 1-38, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is associated with a phenotypic trait / characteristic 42. The method of embodiment 41, the method further comprising determining a phenotypic trait of the fetus, wherein:i) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the phenotypic trait, orii) the absence of a target nucleic acid is detected and the fetus is determined not to have or likely develop the phenotypic trait.43. A method of detecting the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of embodiments 1-38, wherein the biological sample is plasma separated from a maternal whole blood sample,wherein the nucleic acid is a fetal cell-free nucleic acid, andwherein the target nucleic acid is associated with a congenital disease.44. The method of embodiment 43, wherein the method further comprising detecting congenital disease, whereini) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the congenital disease, orii) the absence of the target nucleic acid is detected and the fetus is determined not to have or likely develop the congenital disease.-6- ACTIVE\1627041887.145. A method of detecting the presence or absence of a target nucleic acid in sample from a subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the subject using the method of any one of embodiments 1-38,wherein the nucleic acid is a tumor cell-derived nucleic acid, andwherein the target nucleic acid is associated with cancer.46. The method of embodiment 45, the method further comprising detecting minimal residual disease (MRD), wherein:i) the presence of the target nucleic acid is detected and the subject is determined to have MRD, orii) the absence of the target nucleic acid is detected and the subject is determined to not have MRD.47. The method of any one of embodiments 1-46, wherein the sample is from a human subject.48. The method of any one of embodiments 1-47, wherein the method is an extraction-free method.49. The method of any one of embodiments 1-48, wherein the method does not comprise extracting, isolating and / or purifying the target nucleic acid from the sample prior to step c), optionally for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to step c).BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIGs. 1A-B show embodiments of quantification cycle (Cq) value separation based on fetal sex with male samples represented with diamonds and female samples represented by circles. FIG. 1A shows Cq values from plasma samples tested with a commercially-available assay which includes a DNA extraction step and FIG. IB shows Cq values from plasma samples tested with an embodiment of an extraction-free assay.

[0005] FIGs. 2A-B show embodiments of qPCR amplification plots of male fetus samples #1, #2, #3, #4 tested with the commercially-available assay which includes a DNA extraction step (FIG. 2A) and an embodiment of an extraction-free assay (FIG. 2B).-7- ACTIVE\1627041887.1

[0006] FIGs. 3A-B show an embodiment of the qPCR application plots of female fetus samples #11. #12, #13 tested with the commercially-available assay which includes a DNA extraction step (FIG. 3A) and an embodiment of an extraction-free assay (FIG. 3B).

[0007] FIG. 4 shows an embodiment of a picture of a gel electrophoresis with the purified qPCR products from an embodiment of an extraction-free assay. The lane assignments correspond to M: Molecular weight marker; lane 1: Male Sample #1 - Replicate 1. lane 2: Male Sample #2 - Replicate 1, lane 3: Male Sample #3 - Replicate 1, lane 4: Male Sample #4 -Replicate 1, lane 5: Female Sample #11 - Replicate 1, lane 6: Female Sample #12 - Replicate 1, lane 7: Female Sample #13 - Replicate 1, lane 8: negative control, lane 9: positive control, lane 10: non-template control. The gel electrophoresis results confirm the amplification of the target Y-chromosome sequence.DETAILED DESCRIPTION

[0008] The present disclosure includes embodiments of methods and assays for nucleic acid detection in biological samples that forego DNA isolation and purification. Traditional approaches to nucleic acid detection that include DNA isolation / extract! on and / or purification often necessitated large sample volumes, leading to discomfort for patients during sample collection and logistical challenges in sample storage and transportation. Moreover, these conventional methods generated substantial biohazard waste and faced issues such as genomic DNA carryover from cell components, contamination, and inconsistency in results. The interference of PCR inhibitors, often introduced during the isolation and / or purification process, further complicated the analysis by reducing DNA yield and integrity, and by limiting the comparability of circulating free DNA (cfDNA) values from different studies. Additionally, the requirement for expensive instrumentation increased the complexity and cost of these procedures.

[0009] In contrast, some embodiments of the methods and assays described herein simplify the entire process of nucleic acid detection by eliminating the need for DNA isolation, purification, and / or expensive instrumentation. In some embodiments, these approaches significantly reduce the risks of genomic DNA carryover, contamination, and inconsistency. In some embodiments, the elimination of purification steps also removes the risk of PCR inhibitor carryover from the isolation and / or purification reagents, thereby enhancing assay resolution and ensuring more accurate quantitative and qualitative outcomes. In some embodiments, streamlined processes not only shorten processing times but also improve DNA yield and integrity, and in some embodiments allow for universal comparability of cfDNA values across different studies. Importantly, in some embodiments, the removal of the need for expensive -8- ACTIVE\1627041887.1instrumentation renders the methods and assays more accessible and cost-effective for a wide range of applications in both research and clinical settings.

[0010] In some embodiments, the nucleic methods and assays disclosed herein provide solutions to one or more of the limitations inherent in traditional methods that include a DNA isolation / extraction and / or purification step. In some embodiments, by completely removing DNA isolation / extraction and purification steps while enhancing efficiency, accuracy, and / or cost-effectiveness, the novel methods and assays disclosed herein advance the field of molecular diagnostics. The embodiments of the methods and assays disclosed herein can be utilized in a variety of biomedical research and clinical applications.Terms

[0011] Unless expressly stated otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art read in light of the entire specification. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise.

[0012] As used herein, the term "about." has its plain and ordinary meaning as read in light of this disclosure, and refers to a quantity, value, number, frequency, percentage, amount, weight, concentration, or volume that varies + / - 10% to a reference quantity, value, number, frequency, percentage, amount, weight, concentration, or volume.

[0013] As used herein interchangeably, the terms “subject,’’ “individual,” or “patient,” have their plain and ordinary meanings as read in light of this disclosure, and generally refer to a vertebrate, optionally a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.Extraction-Free Assay

[0014] In some embodiments of the methods disclosed herein, detection of the presence or absence of the target nucleic acid is performed using all or an aliquot of the biological sample. In some embodiments, the target nucleic acid is not extracted, isolated, and / or purified from the biological sample prior to the detection step. In some embodiments, the biological sample is plasma, and except for the separation of the plasma sample from a whole blood sample, the target nucleic acid is not extracted, isolated, and / or purified from the-9- ACTIVE\1627041887.1biological sample prior to the detection step. In some embodiments, the target nucleic acid of the biological sample are not extracted, isolated, and / or purified from the biological sample using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step. These embodiments are referred to herein as an “extraction-free'’ method or assay.Biological Sample

[0015] Provided herein are methods of detecting the presence or absence of a target nucleic acid (e.g., cell-free DNA) in a biological sample. In some embodiments, a biological sample including a target nucleic acid is obtained from a subject. In some embodiments, the subject is a mammal subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is apregnant subject. In some embodiments, the subject is apregnant human subject.

[0016] In some embodiments, the biological sample obtained from the subject is typically whole blood or plasma separated from whole blood, but the sample can be any sample from bodily fluids, tissue or cells comprising the nucleic acids to be detected. In some embodiments, the biological sample is selected from, but is not limited to, whole blood, plasma, serum, saliva, urine, cervical mucus and / or amniotic fluid. In some embodiments, the biological sample is obtained through blood collection, urine collection, a cervical swab, a buccal swab, biopsy and / or amniocentesis.

[0017] In some embodiments, the biological sample is blood. In some embodiments, the blood is venous blood. In some embodiments, the blood is obtained from any suitable area of the body, including an arm, a leg, a finger, or blood accessible through a central venous catheter. In some embodiments, blood is collected from the arm via venipuncture. In some embodiments, the blood is not venous blood. In some embodiments, the blood is capillary blood. In some embodiments, capillary blood is collected from the finger, optionally using a lancet. In some embodiments, the capillary blood is blotted and dried on filter paper. In some embodiments, capillary blood is collected using a blood collection device that utilizes microneedles and negative pressure to draw blood into a collection tube (e.g., a TAP device (Seventh Sense Biosystems, MA) or a Tasso-SST device (Tasso Inc., WA)). In some embodiments, the site of blood collection is a site less likely to be contaminated with foreign nucleic acids, such as foreign DNA. In some embodiments, the site of blood collection is on the torso (e.g. stomach, side, back, shoulder), hip, upper leg (e.g. thigh), lower leg (e.g.-10- ACTIVE\1627041887.1calf). In some embodiments, the site of blood collection is the upper arm (i.e., located between the shoulder joint and elbowjoint). In some embodiments, the blood is capillary blood collected from the upper arm using a blood collection device that utilizes microneedles and negative pressure to draw blood into a collection tube (e.g., a TAP device (Seventh Sense Biosystems, MA) or a Tasso-SST device (Tasso Inc., WA)). In some embodiments, the site of blood collection is not the finger, or not the hand. In some embodiments, blood is collected following a treatment or activity. For example, blood can be collected following a medical exam of the subject. In some embodiments, the timing of collection can also be coordinated to increase the amount of target nucleic acids, such as target cell-free nucleic acids, present in the sample. In some embodiments, blood can be collected from the subject following physical exercise.

[0018] In some embodiments, blood is combined with various components following collection to preserve or prepare samples for subsequent techniques. For example, in some embodiments, blood is treated with an anticoagulant, a cell fixative, a protease inhibitor, a phosphatase inhibitor, a protein, a DNA, and / or an RNA preservative following collection. In some embodiments, the biological sample is incubated with a preservative. In some embodiments, the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), heparin), an antimicrobial (e.g., imidazolidinyl urea), a sugar, and / or an amino acid. In some embodiments, the preservative is a solid, a liquid, and / or a gel. In some embodiments, blood is collected via venipuncture using vacuum collection tubes containing an anticoagulant such as EDTA, EGTA, and / or heparin. In some embodiments, blood is collected using a heparin-coated syringe and hypodermic needle. In some embodiments, blood is combined with reagents that will be useful for subsequent detection of the target nucleic acids contained therein.

[0019] In some embodiments, the biological sample volume obtained from the subject is, is about, is not more than, or is not less than 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 pL, or a range defined by any two of the preceding values, for example, 0.25-50, 0.25-100, 2-50, 2-100. 0.25-500, 1-500, 2-500, 5-500, 10-500, 100-400, 1-100, 0.25-100, 400-1000, 1-5000 or 100-2000 pL. In some embodiments, the biological sample volume used to detect the target nucleic acid is a microvolume. In some embodiments, the microvolume is about is, is about, is not more than, or is not less than 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pL, or a range defined by any two of the preceding values, for example, 0.25-500. 1-500, 2-500, 5-500. 10-500, 100-400, 1-100, 0.25-100, 400-1000, 0.25-50, 5-50, 50-100, 5-20, 5-15, 0.25-20, 0.25-15, 1-12, or 0.25- -11- ACTIVE\1627041887.110 pL. In some embodiments, the biological sample volume is less than about 500 pL. In some embodiments, the biological sample volume is less than about 100 pL. In some embodiments, the biological sample volume is 5-15 pL, optionally 10 pL.

[0020] In some embodiments, biological samples undergo mechanical manipulation including centrifugation, sedimentation, and / or fdtration. In some embodiments, the biological sample is plasma. In some embodiments, plasma is separated from a whole blood sample using centrifugation, sedimentation, and / or filtration (e.g., utilizing membranes and / or microfluidic devices). In some embodiments, the plasma is separated from a capillary blood sample. In some embodiments, the plasma is separated from a capillary blood sample using centrifugation.

[0021] In some embodiments, the plasma sample volume is, is about, is not more than, or is not less than 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 pL, or a range defined by any two of the preceding values, for example, 0.2-100, 1-100, 1-50, 50-100, 1-25, 0.2-10, 5-15, or 0.2-5 pL. In some embodiments, the whole blood sample volume from which the plasma sample is obtained is, is about, is not more than, or is not less than 2.5, 5. 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100. 200, 300, 400, or 500 pL, or a range defined by any two of the preceding values, for example, 2.5-500, 2.5-100, 2.5-50, 50-100, 20-50, or 2.5-10 pL. In some embodiments, the plasma sample volume is about 1-100 pL, and the whole blood sample volume is about 2.5-500 pL. In some embodiments, the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL. In some embodiments, the plasma sample volume is about 2 pL, and the whole blood sample volume is about 5 pL.Target Nucleic Acids

[0022] In some embodiments, the target nucleic acid in the biological sample is DNA or RNA. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is a genomic nucleic acid of the subject. In some embodiments, the target nucleic acid is a tumor cell-derived nucleic acid of the subject. In some embodiments, the biological sample is from a pregnant subject and the target nucleic acid is a fetal nucleic acid.

[0023] In some embodiments, the portion of the target nucleic acid amplified for detection ranges in length from about 15-500 nucleic acids. In some embodiments, the portion of the target nucleic acid amplified for detection, is, is about, is not more than, or is not less than 15, 20, 25, 50, 75, 100, 125, 150, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,-12- ACTIVE\1627041887.1475 or 500 nucleotides, or a range defined by any two of the preceding values, for example 15-500, 15-250, 15-100. 100-500. 200-400. 50-150, 50-250, 15-50 nucleotides in length. In some embodiments, the portion of the target nucleic acid amplified for detection is about 50-250 nucleotides in length. In some embodiments, the portion of the target nucleic acid amplified for detection is about 50-150 nucleotides in length.

[0024] In some embodiments, the amount of target nucleic acid within in the biological sample (e.g., whole blood or plasma) is quantified as a concentration of target nucleic acid per sample volume (for example, measured in ng / mL), a number of copies of target nucleic acid per sample volume (for example, measured in copies / pL), or a number of genome equivalents (GE) per sample volume (for example, measured in GE / mL), or as a percentage of the total amount or of a subset of nucleic acids in the biological sample.

[0025] In some embodiments, the biological sample (e.g., whole blood or plasma) includes a concentration of target nucleic acid per sample volume (ng / mL). In some embodiments, the biological sample includes a concentration of target nucleic acid per sample volume, wherein the concentration of target nucleic acid per sample volume is, is about, is not more than, or is not less than about 0.0003, 0.0006. 0.001, 0.003. 0.006, 0.01, 0.03, 0.06, 0.1.0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any two of the preceding values, for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, or 0.01-3 ng / mL. In some embodiments, the biological sample includes a concentration of target nucleic acid per sample volume, wherein the concentration of target nucleic acid per sample volume is 0.03-2 ng / mL, optionally 0.5 ng / mL.

[0026] In some embodiments, the number of copies of the target nucleic acid per sample in the biological sample (e g., whole blood or plasma) is, is about, is not more than, or is not less than, 0.1, 0.5, 1, 5, 10, 50. 100, 200, 300, 400, 500. 600, 700, 800, 900, 1000, 5000, 10000, or 15000 copies of the target nucleic acid per sample volume (copies / pL) or a range defined by any two of the preceding values, for example 0.1-15000, 1-5000, 1-1000, 5-10000, 5000-15000, 10-5000, 1-200, or 1-50 copies of the target nucleic acid per sample volume (copies / pL). In some embodiments, the copies of the target nucleic acid per sample volume (copies / pL) is 15-3000 copies of the target nucleic acid per sample volume (copies / pL), optionally about 300 copies of the target nucleic acid per sample volume (copies / pL).

[0027] In some embodiments, the amount of nucleic acid in the sample (e.g., whole blood or plasma) is quantified using genome equivalents. A genome equivalent is the amount of DNA contained in a single genome, and the amount of DNA contained in a single genome can be measured in grams (e.g., pg) or bases (e.g., Mb). The size of one genome equivalent is -13- ACTIVE\1627041887.1specific to individual species, and whether the genome of interest is a haploid genome or diploid genome. In some embodiments, genome equivalents are calculated as human genome equivalents. In some embodiments, genome equivalents are calculated as haploid genome equivalents. In some embodiment, genome equivalents are calculated as diploid genome equivalents. In some embodiments, genome equivalents are calculated as human haploid genome equivalents. In some embodiments, genome equivalents are calculated as human diploid genome equivalents. Unless specified otherwise, genome equivalents (GE) used herein are human haploid genome equivalents. In some embodiments, the GE in the biological sample is, is about, is not more than, is not less than, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 1500, 2000, 2500 or 3000 GE of target nucleic acid / mL, or a range defined by any two of the preceding values, for example 0.1-3000. 0.1-1000, 0.1-500, 500-1000, 500-2500. 1-3000, 0.1-100, 0.1-50, 1-50, 0.1-25, 0.1-10, 1-10, 0.1-5, 0.1-2.5, 0.1-1 GE of target nucleic acid / mL. In some embodiments, the concentration of the target nucleic acid in the biological sample is, or is less than, 3000, 2500, 2000, 1500, 1000, 500, 250, 100, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 GE / mL of biological sample, or a range defined by any two of the preceding values, for example 0.1-500, 0.1-100, 0.1-50, 1-10, 1-5, 0.5-10, 0.5-5 GE / ml of biological sample. In some embodiments, the biological sample includes 10-500 GE of target nucleic acid per mL of sample. In some embodiments, the biological sample includes 10-500 GE of target nucleic acid per mL of whole blood sample. In some embodiments, the biological sample includes 10-500 GE of target nucleic acid per mL of plasma sample.

[0028] In some embodiments, the target nucleic acid is a cell-free nucleic acid, optionally a cell-free DNA (cfDNA) or a cell-free RNA (cfRNA). In some embodiments, the target nucleic acid is a cell-free DNA (cfDNA). In some embodiments, the target nucleic acid is a cell-free DNA (cfRNA). In some embodiments, the target nucleic acid is a genomic cfDNA of the subject. In some embodiments, the target nucleic acid is a tumor cell-derived cfDNA of the subject. In some embodiments, the biological sample is from a pregnant subject and the target nucleic acid is a fetal cfDNA.

[0029] In some embodiments, the biological sample (e.g., whole blood or plasma) includes a concentration of target cell-free (cl) nucleic acid (e.g.. cfDNA) per sample volume (ng / mL). In some embodiments, the biological sample includes a concentration of target cf nucleic acid (e.g., cfDNA) per sample volume, wherein the concentration of target cf nucleic acid per sample volume is, is about, is not more than, or is not less than about 0.0003, 0.0006, 0.001, 0.003. 0.006, 0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8. 0.9, 1, 1.1. 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any two of -14- ACTIVE\1627041887.1the preceding values, for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, or 0.01-3 ng / mL. In some embodiments, the biological sample includes a concentration of target cf nucleic acid (e.g., cfDNA) per sample volume, wherein the concentration of target nucleic acid per sample volume is 0.03-2 ng / mL, optionally 0.5 ng / mL.

[0030] In some embodiments, the number of copies of the target cf nucleic acid (e.g., cfDNA) per sample in the biological sample (e.g., whole blood or plasma) is, is about, is not more than, or is not less than, 0.1, 0.5, 1. 5, 10, 50, 100, 200, 300. 400, 500. 600, 700, 800, 900, 1000, 5000, 10000, or 15000 copies of the target cf nucleic acid per sample volume (copies / pL) or a range defined by any two of the preceding values, for example 0.1-15000, 1-5000, 1-1000, 5-10000, 5000-15000, 10-5000, 1-200, or 1-50 copies of the target nucleic acid per sample volume (copies / pL). In some embodiments, the copies of the target cf nucleic acid (e.g., cfDNA) per sample volume (copies / pL) is 15-3000 copies of the target nucleic acid per sample volume (copies / pL), optionally about 300 copies of the target cf nucleic acid per sample volume (copies / pL).

[0031] In some embodiments, the GE of the target cf nucleic acid (e.g., cfDNA) in the biological sample is, is about, is not more than, is not less than, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 1500, 2000, 2500 or 3000 GE of target nucleic acid / mL, or a range defined by any two of the preceding values, for example 0.1-3000, 0.1-1000, 0.1-500, 500-1000, 500-2500, 1-3000. 0.1-100, 0.1-50, 1-50, 0.1-25, 0.1-10, 1-10, 0.1-5, 0.1-2.5, 0.1-1 GE of target cf nucleic acid / mL. In some embodiments, the biological sample includes 10-500 GE of target nucleic cf acid (e.g., cfDNA) per mL of sample. In some embodiments, the biological sample includes 10-500 GE of target nucleic acid per mL of whole blood sample. In some embodiments, the biological sample includes 10-500 GE of target cf nucleic acid (e.g., cfDNA) per mL of plasma sample.

[0032] In some embodiments, the target cf nucleic acid (e.g., fetal cfDNA) is quantified as a percentage of a subset of total nucleic acids in the biological sample, for example, the percentage of target cf nucleic acids of the total amount of cell-free nucleic acids in the biological sample. In some embodiments, the target cf nucleic acid (e.g., cfDNA) comprises about 1. 2, 3, 5, 6. 7, 8, 9. 10. 11, 12, 13, 14, or 15%. or a range defined by any two of the preceding values, for example, 1-15, 1-12, 5-10, or 2-10% of the total amount of cf nucleic acid (e.g., cfDNA) in the biological sample. In some embodiments, the target cf nucleic acid (e.g., cfDNA) comprises 2-10% of the total amount of cfDNA in the biological sample. In some embodiments, the target nucleic acid is fetal cfDNA and comprises 2-10% of the total amount of cfDNA in the biological sample from the pregnant subject.-15- ACTIVE\1627041887.1

[0033] In some embodiments, the target nucleic acid in the biological sample is located on one or more chromosomes. In some embodiments, the target nucleic acid is located only on the Y-chromosome. In some embodiments, the target nucleic acid is present in multiple locations in the genome such that there are multiple copies per genome. In some embodiments, the target nucleic acid is present as, as about, or as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500. 1000, 2000, 3000. 4000, 5000, 6000. 7000, 8000, 9000, or 10000 copies per genome, or a range defined by any two of the preceding values, for example 1-10000, 1-6000, 4000-10000, 2000-8000, 1-500, 1-100, 1-10, 10-100, or 1-50 copies per genome. In some embodiments, the target nucleic acid is present as 2000-8000 copies per genome. In some embodiments, the target nucleic acid is present as 1-10 copies per genome. In some embodiments, the target nucleic acid is present on only the Y-chromosome in multiple locations such that there are multiple copies per Y-chromosome. In some embodiments, the target nucleic acid is present as, as about, as not less than, or as not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 copies on the Y-chromosome, or a range defined by any two of the preceding values, for example 1-10000. 1-6000, 4000-10000, 2000-8000, 1-500. 1-100.1-10, 10-100, or 1-50 copies per Y-chromosome. In some embodiments, the target nucleic acid is present as 2000-8000, or 4500-6000 copies per Y-chromosome. In some embodiments, the target nucleic acid is present as 1-10 copies per Y-chromosome, optionally 4-10 copies per Y-chromosome. In some embodiments, the target nucleic acid on the Y-chromosome is DAZ (Deleted in Azoospermia), DAZ1, DAZ2, DAZ3, DAZ4, TSPY (Testis-Specific Protein Y-encoded), TSPY2, TSPY3, TSPY4, RBMY (RNA-Binding Motif Y), BPY2 (Basic Protein Y-2), PRY (Pseudogene on the Y), DYZ1 (Alphoid Repeats on Y), DYZ2 (Repetitive Sequence on Y), DYZ3 (Additional Alphoid Repeats on Y), PRY2, HSFY (Heat Shock Factor Y), V CY (Variable Charge Y), VCY2, CDY (Chromodomain Protein Y), CDY1, CDY2, XKRY (XK-Related Y), TTTY (Testis-Specific Transcript Y). In some embodiments, the target nucleic acid on the Y-chromosome is SRY, DYS, and / or DAZ.

[0034] In some embodiments, the target nucleic acid is associated with a phenotypic trait. A phenotypic trait is an observable characteristic of a subject. In some embodiments, the phenotypic trait is selected from, but is not limited to, eye color, hair color, hair texture, blood type, Rh factor, presence of freckles, presence of cheek dimples, and / or adult height. In some embodiments, the phenotypic trait is Rh factor. In some embodiments, the target nucleic acid is associated with a phenotypic trait. In some embodiments, the target-16- ACTIVE\1627041887.1nucleic acid includes a polynucleotide sequence including all or a portion of a target gene. In some embodiments, the target gene is RHD, HERC2, OCA2, MC1R, or GHR.

[0035] In some embodiments, the target nucleic acid is associated with a pathological condition, such as a congenital disease, a genetic disorder, and / or a cancer. In some embodiments, the pathological condition is selected from, but is not limited to, Down syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), Patau syndrome (Trisomy 13), 22ql l.2 Deletion Syndrome (DiGeorge Syndrome), or Turner syndrome (Monosomy X). In some embodiments, the congenital disease is congenital heart defect. In some embodiments, the target nucleic acid is associated with a congenital disease, optionally a mutation of a target gene associated with a congenital disease. In some embodiments, the target gene associated with a congenital disease is TBX1, GATA4, or NKX2-5. In some embodiments, the genetic disorder is selected from, but is not limited to, cystic fibrosis, sickle cell disease, betathalassemia, hemophilia A, or spinal muscular atrophy (SMA). In some embodiments, the genetic disorder is cystic fibrosis. In some embodiments, the target nucleic acid is associated with a genetic disorder, optionally a mutation of a target gene associated with a genetic disorder. In some embodiments, the target gene associated with a genetic disorder is CFE' R, HBB, F8, or SMNJ.

[0036] Cancers are diseases characterized by the development of abnormal cells that divide uncontrollably and have the potential to spread throughout different parts of the body. Cancers are often associated with genetic mutations. In some embodiments, genetic mutations are inherited gene mutations. In some embodiments, genetic mutations are acquired gene mutations. In some embodiments, the target nucleic acid is associated with a cancer. In some embodiments, the target nucleic acid includes a polynucleotide sequence including all or a portion of a mutation of a target gene associated with cancer. In some embodiments, the target gene is a proto-oncogene, tumor suppressor gene, or DNA repair gene. In some embodiments, the target gene associated with cancer is TP53, KRAS, EGFR, BRCA1, BRCA2, MYC, or ARC.Treatment of the Biological Sample

[0037] In some embodiments of the disclosed methods, the biological sample is treated prior to detection of the target nucleic acid. In some embodiments, a treatment is applied to a biological sample to release nucleic acids bound to proteins in the biological sample. In some embodiments, a treatment is applied to a biological sample to reduce interference by substances present in the sample with detection of the target nucleic acid, for example proteins in the sample interfering with amplification of the target nucleic acid. In some embodiments, a -17- ACTIVE\1627041887.1treatment is applied to a biological sample to release nucleic acids bound to proteins in the biological sample and to reduce interference by substances present in the sample with detection of the target nucleic acid, for example proteins in the sample interfering with amplification of the target nucleic acid. In some embodiments, the treatment is a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the biological sample is incubated at ambient / room temperature during the application of the treatment.

[0038] In some embodiments, the method includes an additional step to activate the treatment after it is applied to the biological sample. In some embodiments, the treatment (e.g., proteinase K) is activated in order to release nucleic acids bound to proteins and to degrade proteins present in the biological sample that are PCR inhibitors. In some embodiments, activation of the treatment (e.g., proteinase K) after it is applied to the biological sample includes incubation of the biological sample at an activation temperature for a period of time. In some embodiments, the activation temperature is, is about, is not more than, or is not less than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 °C, or a range defined by any two of the preceding values, for example, 20-42, 27-42, 27-40, 30-42, 30-40, or 35-40 °C. In some embodiments, the activation temperature (e.g., of proteinase K) is 37 °C. In some embodiments, the period of time for treatment (e g., proteinase K) activation is, is, is about, is not more than, or is not less than, 10, 15, 20, 25, 30, 35, 45, 50 minutes, or a range defined by any two of the preceding values, for example, 10-50, 10-40, 20-50, 20-40, or 25-35 minutes. In some embodiments, the period of time for treatment (e.g., proteinase K) activation is 30 minutes.

[0039] In some embodiments of the disclosed methods, the treatment of the biological sample is terminated. In some embodiments, terminating the treatment is inactivation of the treatment. In some embodiments, terminating the treatment is inactivation of the proteinase. In some embodiments, terminating the treatment is a chemical inactivation of the treatment. In some embodiments, terminating the treatment is thermal inactivation of the treatment. In some embodiments, terminating the treatment is thermal inactivation of the proteinase (e.g., proteinase K). In some embodiments, inactivation of the treatment (e.g., proteinase K) includes incubation of the biological sample at an inactivation temperature for a period of time. In some embodiments, the inactivation temperature is, is about, is not more than, or is not less than, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 °C, or a range defined by any two of the preceding values, for example, 45-65, 45-60, 50-65, 50-60, or 52-57 °C. In some embodiments, the inactivation temperature (e.g.. of proteinase K) is 55 °C. In some embodiments, the inactivation period of time is is, is about, is not more than, or is not -18- ACTIVE\1627041887.1less than, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 minutes,, or a range defined by any two of the preceding values, for example, 5-25, 5-20, 10-25, 5-15, or 8-12 minutes. In some embodiments, the inactivation period of time is 10 minutes.

[0040] In some embodiments, the treatment further comprises at least one chemical reagent applied to the biological sample. In some embodiments, the at least one chemical reagent is a detergent, optionally the detergent is sodium dodecyl sulfate (SDS). In some embodiments, the detergent is applied to / incubated with the biological sample for a shorter, equal or longer duration of time than the remainder of the treatment (e.g., proteinase K) is applied to or incubated with the biological sample. In some embodiments, the detergent (e.g., SDS) is applied to the biological sample at a concentration that is, is about, is not more than, is not less than. 0.001, 0.002. 0.005, 0.01, 0.02. 0.05, 0.1, 0.2. 0.5, 1, 2, 5. 10. 12. 15. or 20%, or a range defined by any two of the preceding values, for example, 0.001-20, 0.001-1. 0.002-2, 0.05-10. 0.1-10, or 0.002-0.5%. In some embodiments, the detergent (e.g., SDS) is applied to the biological sample at a concentration that is 0.005-0.2%, optionally 0.02%.

[0041] In some embodiments, the biological sample is incubated at ambient / room temperature after the termination of the treatment.

[0042] In some embodiments, the biological sample is diluted prior to performing an assay (e.g., qPCR) to detect the presence or absence of the target nucleic acid in the biological sample. In some embodiments, the sample is diluted prior to treatment (e.g., proteinase K treatment) of the sample as described above and elsewhere herein. In some embodiments, the sample is diluted after termination of treatment (e.g., proteinase K treatment) of the sample as described above and elsewhere herein. In some embodiments, when measured upon termination of the treatment (e.g., proteinase K treatment) of the biological sample (e.g., plasma), the biological sample is diluted not more than 1:40 (sample: total volume). In some embodiments, when measured upon termination of the treatment (e.g., proteinase K treatment) of the biological sample (e.g., plasma), the biological sample is diluted to: 1:1.25 to 1:35, 1:2 to 1:20, or 1:5 to 1:10. In some embodiments, when measured upon termination of the treatment (e.g., proteinase K treatment) of the biological sample (e.g., plasma), the biological sample is diluted to about 1:1.25, 1:1.5, 1:2. 1:4, 1:5, 1:6. 1:7, 1:8, 1:9. 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, or a range defined by any two of the preceding values. In some embodiments, when measured upon termination of the treatment (e.g., proteinase K treatment) of the biological sample (e.g., plasma), the biological sample is diluted to about 1:10.-19- ACTIVE\1627041887.1Detecting the Target Nucleic Acid

[0043] In some embodiments of the disclosed methods, nucleic acid detection assays are utilized to detect the presence or absence of a target nucleic acid in a biological sample. In some embodiments, the detection assay is selected from, but is not limited to, quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR), isothermal nucleic acid amplification, nested polymerase chain reaction, polymerase chain reaction (PCR) followed by gel electrophoresis, and nucleic acid sequencing. In some embodiments, the detection assay is qPCR. In some embodiments, qPCR is singleplex qPCR, duplex qPCR, or multiplex qPCR. In some embodiments, qPCR is multiplex qPCR. In some embodiments, qPCR to detect the presence or absence of the target nucleic acid in the biological sample is performed using all or an aliquot of the biological sample. In some embodiments, the qPCR reaction includes a master mix and all or an aliquot of the biological sample.

[0044] In some embodiments, the master mix used in the qPCR includes a reaction mixture including at least one polymerase and deoxynucleotide triphosphates (dNTPs). In some embodiments, the concentration of dNTPs is, is about, is not more than, or is not less than 1, 1.5, 2, 5, 7.5, 10, 15, 20, 25, 30, 40 or 50 mM dNTPs, or a range defined by any two of the preceding values, for example, 1-50, 1-10, 25-50, or 5-25 mM dNTPs. In some embodiments, the dNTPs is ultrapure dNTPs. In some embodiments, the dNTPs comprise dATP, dGTP, dCTP, dTTP, dUTP, or any combination thereof.

[0045] In some embodiments, the at least one polymerase is a genetically modified polymerase. In some embodiments, the at least one polymerase has a high tolerance to common PCR inhibitors. Common PCR inhibitors are factors that disrupt and prevent nucleic acid amplification during PCR, and can include substances present in biological samples such as heme, hemoglobin, lactofenin, immunoglobin G (IgG), and urea; ionic detergents including SDS, sodium deocy cholate, and sarkosyl; organic compounds including ethanol, isopropanol, and phenol; as well as excess salts including potassium chloride (KC1) and sodium chloride (NaCl). PCR inhibitors typically disrupt amplification by interference with the DNA polymerase or through interaction with the template DNA.

[0046] In some embodiments, use of a polymerase with a high tolerance to common PCR inhibitors is important to extraction-free methods disclosed herein, because the target nucleic acid is able to be directly amplified and detected (e.g., with qPCR) using all or an aliquot of the original biological sample without a nucleic acid isolation, extraction and / or purification step (excepting isolation of plasma from whole blood when the biological sample is plasma). In some embodiments, using a polymerase with a high tolerance to common PCR -20- ACTIVE\1627041887.1inhibitors allows for detergents, such as SDS, to be applied to the biological sample in addition to the treatment (e.g., proteinase K treatment). SDS is an anionic strong detergent that gives net negative charge to the proteins (for example in SDS-PAGE gel electrophoresis), but does not influence the negatively charged DNA during the treatment. In some embodiments, the SDS lyses cells present in the biological sample and / or denatures proteins in the sample (e.g., by irreversibly destroying their three-dimensional (3D) structures), but does not denature polymerases with a high tolerance to common PCR inhibitors. In some embodiments, the at least one polymerase has a high inhibitory tolerance to SDS.

[0047] In some embodiments, the at least one polymerase is a hot start thermostable polymerase. Hot start thermostable polymerases can be utilized in hot start PCR methods. Hot start PCR is a modified form of conventional polymerase chain reaction (PCR). Hot start PCR typically involves the use of a polymerase that is inactivated at lower and ambient temperatures, and that is subsequently activated at higher temperatures, usually during the denaturation step of PCR (e.g., when the reaction reaches a temperature of 90-105° C., e.g., 95° C). In some embodiments, the qPCR reaction including the hot start thermostable polymerase is incubated at a specific temperature of or of about 85, 90, 95, 100. 105, or 110° C or a range defined by any two of the preceding values, for example, 85-110, 90-110, or 90-100 ° C. In some embodiments, the qPCR reaction including the hot start thermostable polymerase is incubated for a period of time of or of about 1, 2, 3, 4, 5, 7, 10, 15, 20, or 30 minutes, or a range defined by any two of the preceding values, for example, 1-30, 1-10, or 2-5 minutes. For example, the qPCR reaction is incubated for 3 minutes at 95° C. in order to activate a hot start PCR polymerase. In some embodiments, the use of such a hot start thermostable polymerase prevents extension of non-specifically annealed primers and primer-dimers formed at low- temperatures during PCR setup. In some embodiments, the hot start PCR technique avoids nonspecific amplification of DNA, and increases the sensitivity and yield of the PCR reaction. Many hot-start thermostable polymerases are antibody-based, which do not tolerate SDS and are unable to be used to amplify unpurified target nucleic acids in biological samples where SDS is added. In some embodiments, selecting a hot-start thermostable polymerase that has a high inhibitory tolerance to SDS allows for the target nucleic acid to be directly amplified and detected with fast-activated qPCR using all or an aliquot of the original biological sample where SDS is added. In some embodiments, the at least one polymerase is a hot-start thermostable polymerase that has a high inhibitory tolerance to SDS.

[0048] In some embodiments, the master mix includes magnesium chloride (MgCl₂). In some embodiments, the initial concentration of MgCl₂ added to the master mix is,-21- ACTIVE\1627041887.1is about, is not more than, or is not less than, 0.1, 0.2, 0.5, 1, 2, 5, or 10 M or a range defined by any two of the preceding values, for example. 0.1-10, 0.1-2, 0.1-5, 2-10, or 0.5-2 M. In some embodiments, the initial concentration of MgCl₂ added to the master mix is 1 M. In some embodiments, the volume of MgCl₂ added to the master mix is, is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1, or 0.05-0.5 pL. In some embodiments, the volume of MgCl₂ added to the master mix is 0.0625 pL. In some embodiments, the master mix includes nuclease free water. In some embodiments, the volume of nuclease free water added to the master mix is about, is not more than, or is not less than, 0.01, 0.002, 0.05. 0.1, 0.2, 0.5, 1, or 2 pL or a range defined by any two of the preceding values, for example, 0.01-2, 0.01-0.2, 0.01-0.5, 0.02-0.1, 0.05-0.5, 0.05-2 pL. In some embodiments, the volume of nuclease free water added to the master mix is 0.188 pL.

[0049] In some embodiments, the master mix includes forward and reverse primers designed to amplify a control reference gene. In some embodiments, for qPCR applications, a housekeeping gene is selected as the control reference gene. Housekeeping genes are typically essential for the maintenance of basic cellular function and are expressed at relatively constant levels across different cell and tissue types. In some embodiments, a control reference gene is used as an internal qPCR reaction control. In some embodiments, a control reference gene is utilized for the relative quantification of target nucleic acid expression and detection. In some embodiments, the control reference gene is an autosomal control reference gene. In some embodiments, the control reference gene is a housekeeping gene. In some embodiments, the control reference gene is 18S ribosomal RNA (RNA18s). In some embodiments, the volume of the forward control reference gene primer added to the master mix is, is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1, or 0.05-0.5 pL. In some embodiments, the volume of the reverse control reference gene primer added to the master mix is, is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1. or 0.05-0.5 pL. In some embodiments, the volume of the forward control reference gene primer added to the master mix is 0.1 pL, and the volume of the reverse control reference gene primer added to the master mix is 0.1 pL.

[0050] In some embodiments, the master mix includes a reporter DNA-binding dye. In some embodiments, the reporter DNA-binding dyes is selected from SYBR® Green,-22- ACTIVE\1627041887.1EvaGreen® dye, LCGreen® dyes, SYTO-9, Chromofy™, and / or BEBO. In some embodiments, the reporter DNA-binding dye is not SYBR® Green dye.

[0051] In some embodiments, the master mix includes at least one qPCR probe. In some embodiments, at least one qPCR probe is designed to amplify all or a portion of a control reference gene. In some embodiments, at least one qPCR probe is designed to amplify all or a portion of a target nucleic acid. In some embodiments, the probe set comprises one or more polynucleotide probes. Individual polynucleotide probes include a nucleotide sequence derived from the nucleotide sequence of the target nucleic acid or complementary sequences thereof In some embodiments, the nucleotide sequence of the polynucleotide probe is designed such that it corresponds to, or is complementary to the target nucleic acid. In some embodiments, the polynucleotide probe can specifically hybridize under either stringent or lowered stringency hybridization conditions to a region of the target nucleic acid. In some embodiments, the selection of the polynucleotide probe sequences and determination of their uniqueness is carried out in silico using techniques known in the art, for example, based on a BLASTN search of the polynucleotide sequence in question against gene sequence databases, such as the Human Genome Sequence, UniGene, dbEST or the non-redundant database at NCBI. Computer programs can also be employed to select probe sequences that do not cross hybridize or do not hybridize non-specifically. In some embodiments, the qPCR probe, is, is about, is not more than, or is not less than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length, or a range defined by any two of the preceding values, for example, 15-100, 50-100, 20-60, or 40-80 nucleotides in length.

[0052] In some embodiments, a label is attached to or incorporated into a probe or primer polynucleotide to allow detection and / or quantitation of a target polynucleotide representing the target sequence of interest. In some embodiments, the qPCR probes are dual-labeled probes. In some embodiments, the dual-labeled probes each include a minor groove-binding moiety. In some embodiments, the dual-labeled probes are TaqMan™ MGB (minor groove binder) probes. In some embodiments, the volume of the at least one qPCR probe is designed to amplify all or a portion of a control reference gene added to the master mix is, is about, is not more than, or is not less than. 0.005. 0.01. 0.02. 0.05. 0.1, 0.2, or 0.5 pL, or a range defined by any two of the preceding values, for example, or 0.005-0.5, 0.02-0.1, 0.1 -0.5, or 0.01-0.1 pL. In some embodiments, the volume of the at least one qPCR probe is designed to amplify all or a portion of a control reference gene is 0.05 pL. In some embodiments, the volume of the at least one qPCR probe is designed to amplify all or a portion of a target nucleic acid is, is about, is not more than, or is not less than, 0.0075, 0.01, 0.02, 0.075, 0.1, 0.2, or 0.75-23- ACTIVE\1627041887.1pL, or a range defined by any two of the preceding values, for example, or 0.0075-0.75, 0.01-0.1, 0.01-0.05, or 0.02-0.2 pL. In some embodiments, the volume of the at least one qPCR probe is designed to amplify all or a portion of a target nucleic acid is 0.075 pL. In some embodiments, the initial concentration of the at least one qPCR probe is designed to amplify all or a portion of a target nucleic acid is, is about, is not more than, or is not less than, 5, 10, 20, 50, 100, 200, or 500 pM, or a range defined by any two of the preceding values, for example, or 5-500, 20-100. 100-500. or 10-100 pM. In some embodiments, the initial concentration of the at least one qPCR probe is designed to amplify all or a portion of a target nucleic acid is 50 pM.

[0053] In some embodiments, the master mix includes forward and reverse primers designed to amplify the target nucleic acid. In some embodiments, the forward and reverse primers designed to amplify the target nucleic acid are, are about, are not more than, or are not less than 15, 16, 17, 18, 19, 20, 25, 50, 100, 150, 200, 250, or 300 nucleotides in length, or a range defined by any two of the preceding values, 15-30, 20-25, 25-300, 50-250, or 100-200 nucleotides in length.

[0054] In some embodiments, the volume of the reverse control reference gene primer added to the master mix is, is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1, or 0.05-0.5 pL. In some embodiments, the volume of the forward control reference gene primer added to the master mix is 0.1 pL, and the volume of the reverse control reference gene primer added to the master mix is 0.1 pL. In some embodiments, the volume of the forward target nucleic acid primer added to the master mix is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1, or 0.05-0.5 pL. In some embodiments, the volume of the reverse target nucleic acid primer added to the master mix is about, is not more than, or is not less than, 0.01, 0.002, 0.05, 0.1, 0.2, 0.5, or 1 pL or a range defined by any two of the preceding values, for example, 0.01-1, 0.01-0.2, 0.01-0.5, 0.02-0.1, or 0.05-0.5 pL. In some embodiments, the volume of the forward target nucleic acid primer added to the master mix is 0.15 pL and the volume of the reverse target nucleic acid primer added to the master mix is 0.15 pL. In some embodiments, the initial concentration of the forward target nucleic acid primer is, is about, is not more than, or is not less than, 5, 10, 20, 50, 100, 200, or 500 pM, or a range defined by any two of the preceding values, for example, or 5-500, 20-100. 100-500. or 10-100 pM. In some embodiments, the initial concentration of the reverse target nucleic acid primer is, is about, is not more than, or -24- ACTIVE\1627041887.1is not less than, 5, 10, 20, 50, 100, 200, or 500 pM, or a range defined by any two of the preceding values, for example, or 5-500, 20-100, 100-500, or 10-100 pM. In some embodiments, the initial concentration of the forward target nucleic acid primer is about 50 pM and the initial concentration of the reverse target nucleic acid primer is about 50 pM.

[0055] In some embodiments, the volume of treated biological sample included in the qPCR reaction is, is about, is not more than, or is not less than 0.1, 0.5, 1, 2. 3, 4, 5, 6, 7, 8, 9, 10. 11. 12. 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23.24. 25.30. 35, 40, 45, 50 pL, or a range defined by any two of the preceding values, for example, 0.1 -30, 0.1 -25, 2-50, 25-50, 2-25, or 10-30, or 20-30 pL. In some embodiments, the volume of treated biological sample included in the qPCR reaction is 21 pL. In some embodiments, the biological sample on which the qPCR is performed is diluted not more than 1:40. In some embodiments, the biological sample on which the qPCR is performed is diluted to 1:1.25 to 1:35, 1:2 to 1:20, or 1:5 to 1:10. In some embodiments, the biological sample on which the qPCR is performed is diluted to about 1: 1.25, 1:1.5, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, or a range defined by any two of the preceding values. In some embodiments, the qPCR reaction volume is, is about, is not more than, or is not less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 pL, or a range defined by any two of the preceding values, for example, 0.1-30, 0.1-25, 2-50, 25-50, 2-30, 10-30 or a 20-30 pL. In some embodiments, the qPCR reaction volume is about 25 pL.

[0056] In some embodiments, the qPCR reaction conditions are set at 3 minutes at 95 °C for the initial denaturation of DNA, followed by 40 cycles of 5 seconds at 95 °C for denaturation and 20 seconds at 60 °C for annealing. In some embodiments, the duration of the annealing phase is, is about, is not more than, or is not less than, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. 95. 100, 105. 110, 115, or 120 seconds, or a range defined by any two of the preceding values, for example, 10-120 seconds, 10-100 seconds, 10-60 seconds, or 30-90 seconds.

[0057] In some embodiments, the methods further include interpreting data generated when detecting the presence of absence of a target nucleic acid in a biological sample. In some embodiments, interpreting data generated when detecting the presence of absence of a target nucleic acid in a biological sample is performed using a machine learning algorithm, a quantitative cycle (Cq) algorithm, or artificial intelligence.-25- ACTIVE\1627041887.1Applications and Use

[0058] In some embodiments, the methods of detecting the presence or absence of a target nucleic acid in a biological sample disclosed herein can be utilized in a variety of applications. In some embodiments, the methods of detecting the presence or absence of a target nucleic acid in a biological sample are utilized in non-diagnostic applications, including, but not limited to, basic research experiments, contamination and quality control testing of donated and / or stored biological samples, genetic ancestry testing, forensic testing, and sample identification. In some embodiments, the methods of detecting the presence or absence of a target nucleic acid in a biological sample are utilized in diagnostic applications.

[0059] In some embodiments, the disclosed methods are used to determine the presence or absence of a target nucleic acid of a fetus of a pregnant subject. In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject includes: detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the any one of the extraction-free methods disclosed herein, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, wherein the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is on the Y chromosome. In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject further includes determining the sex of the fetus, wherein: i) the presence of the target nucleic acid is detected and the fetus is determined to be male, or ii) the absence of a target nucleic acid is detected and the fetus is determined to be female.

[0060] In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject includes: detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the any one of the extraction-free methods disclosed herein, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is associated with a phenotypic trait. In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject further includes determining a phenotypic trait of the fetus, wherein: i) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the phenotypic trait, or ii) the absence of a target nucleic acid is detected and the fetus is determined not to have or likely develop the phenotypic trait.

[0061] In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject includes: detecting the presence or -26- ACTIVE\1627041887.1absence of the target nucleic acid in a biological sample from the pregnant subject using the any one of the extraction-free methods disclosed herein, wherein the biological sample is plasma separated from a maternal whole blood sample, wherein the nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is associated with a congenital disease or genetic disorder. In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject further includes detecting congenital disease or genetic disorder, wherein: i) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the congenital disease or genetic disorder, or ii) the absence of the target nucleic acid is detected and the fetus is determined not to have or likely develop the congenital disease or genetic disorder.

[0062] Disease diagnosis and risk: Also provided are methods of detecting the presence or absence of a target nucleic acid in a biological sample from a subject. In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a sample from a subject includes detecting the presence or absence of the target nucleic acid in a biological sample from the subject using any one of the extraction-free methods disclosed herein, and wherein the target nucleic acid is associated with a disease.

[0063] Cancer method diagnostics: In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a sample from a subject includes detecting the presence or absence of the target nucleic acid in a biological sample from the subject using the any one of the extraction-free methods disclosed herein, wherein the nucleic acid is a tumor cell-derived nucleic acid, and wherein the target nucleic acid is associated with cancer.

[0064] Minimum residual disease (MRD) refers to a small number of cancer cells that remain in a cancer patient subject during treatment or after treatment when a patient is in remission. Detection of MRD can indicate success of treatment and / or cancer recurrence. In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a sample from a subject further includes detecting minimal residual disease (MRD). In some embodiments, the method of detecting minimal residual disease (MRD) includes detecting the presence or absence of the target nucleic acid in a biological sample from the subject using any one of the extraction-free methods disclosed herein, wherein the nucleic acid is a tumor cell-derived nucleic acid, and wherein the target nucleic acid is associated with cancer, wherein: i) the presence of the target nucleic acid is detected and the subject is determined to have MRD, or ii) the absence of the target nucleic acid is detected and the subject is determined to not have MRD.-27- ACTIVE\1627041887.1Assay Performance

[0065] In some embodiments, the methods of the present disclosure are used in assays to detect the presence or absence of a target nucleic acid in a biological sample. In some embodiments, target nucleic acid detection assay performance can be assessed by determining the assay’s sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV), and / or negative predictive value (NPV).

[0066] In some embodiments, the performance of the target nucleic acid detection assay is determined by its sensitivity. In some embodiments, the sensitivity of an assay is, is about, or is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of the preceding values, for example 40-100, 50-90, 60-100, or 90-100%. In some embodiments, the performance of the assay is determined by its specificity. In some embodiments, the specificity of an assay is, is about, or is at least, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of the preceding values, for example 40-100, 50-90, 60-100, or 90-100%. In some embodiments, the performance of the assay is based on area under the ROC curve (AUC). In some embodiments, the AUC of an assay of the disclosure is or is about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95, or a range defined by any two of the preceding values 0.5-0.95, 0.6-0.9, or 0.8-0.95. In some embodiments, the performance of the assay is based on accuracy. In some embodiments, the accuracy of an assay of the present disclosure is or is about 40. 45. 50. 55. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of the preceding values, for example 40-100, 50-90, 60-100, or 90-100%.

[0067] In some embodiments, the performance of the target nucleic acid detection methods of the disclosure have been determined in multiple populations. In some embodiments, the performance of the methods, compositions, and kits of the disclosure have been determined in a population of or of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000 or more subjects, or a range defined by any two of the preceding values, for example 10-1000, 10-100, 500-1000, or 50-300 subjects. In some embodiments, the accuracy of an assay of the disclosure is determined in a population of or of at least 10. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000 or more subjects, or a range defined by any two of the preceding values, for example 10-1000, 10-100, 500-1000, or 50-300 subjects. In some embodiments, the sensitivity of an assay of the disclosure is determined in a population of or of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150. 200, 300, 400. 500, and / or 1,000 or more subjects, or a range defined by any two of the preceding values, for -28- ACTIVE\1627041887.1example 10-1000, 10-100, 500-1000, or 50-300 subjects. In some embodiments, the specificity of an assay of the disclosure is determined in a population of or of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and / or 1,000 or more subjects,, or a range defined by any two of the preceding values, for example 10-1000, 10-100, 500-1000, or 50-300 subjects.Exemplary Embodiments

[0068] In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a biological sample includes a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, and wherein the biological sample volume is less than about 100 pL, optionally wherein the biological sample volume is 1-12 pL or is about 10 pL; b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample, wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step.

[0069] In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a biological sample includes a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, wherein the biological sample is plasma, wherein the method further includes separating the plasma from a whole blood sample using centrifugation, optionally wherein the whole blood sample comprises capillary blood, wherein the plasma sample volume comprises 1-100 pL, and the blood sample volume comprises 2.5-500 pL, optionally wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL; wherein the treatment is a proteinase, optionally proteinase K, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment, wherein step a) further includes applying at least one chemical reagent in addition to the treatment to the biological -29- ACTIVE\1627041887.1sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is SDS (e.g., 0.005-0.2%. optionally 0.02%), wherein step a) further includes activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a period of time, wherein the activation temperature is greater than 20 °C, optionally wherein the activation temperature is about 37 °C, and wherein the period of time is 20-40 minutes or is about 30 minutes; b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume), optionally wherein the biological sample is diluted to about 1:10, wherein terminating the treatment is inactivation of the treatment, wherein is inactivation of the treatment is inactivation of the proteinase, wherein inactivation of the treatment includes incubation of the biological sample at an inactivation temperature for a period of time, wherein the inactivation temperature is less than about 65 °C, optionally wherein the inactivation temperature is about 55 °C, wherein the period of time is 5-15 minutes or is about 10 minutes, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40, and wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step.

[0070] In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject includes: detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method including a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the treatment is a proteinase, optionally proteinase K, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment, wherein step a) further includes applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is SDS (e.g., 0.005-0.2%, optionally 0.02%), wherein step a) further includes activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for -30- ACTIVE\1627041887.1a period of time, wherein the activation temperature is greater than 20 °C, optionally wherein the activation temperature is about 37 °C, and wherein the period of time is 20-40 minutes or is about 30 minutes; b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume), optionally wherein the biological sample is diluted to about 1:10, wherein terminating the treatment is inactivation of the treatment, wherein is inactivation of the treatment is inactivation of the proteinase, wherein inactivation of the treatment includes incubation of the biological sample at an inactivation temperature for a period of time, wherein the inactivation temperature is less than about 65 °C, optionally wherein the inactivation temperature is about 55 °C, wherein the period of time is 5-15 minutes or is about 10 minutes, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40, and wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step; wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, optionally wherein the whole blood sample comprises capillary blood, wherein the plasma sample volume comprises 1-100 pL, and the blood sample volume comprises 2.5-500 pL, optionally wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL, wherein the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is on the Y chromosome; the method further includes determining the sex of the fetus, wherein: i) the presence of the target nucleic acid is detected and the fetus is determined to be male, or ii) the absence of a target nucleic acid is detected and the fetus is determined to be female.

[0071] In some embodiments, the method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject includes: detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method including a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the treatment is a proteinase, optionally proteinase -31- ACTIVE\1627041887.1K, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment, wherein step a) further includes applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is SDS (e.g., 0.005-0.2%, optionally 0.02%), wherein step a) further includes activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a period of time, wherein the activation temperature is greater than 20 °C, optionally wherein the activation temperature is about 37 °C, and wherein the period of time is 20-40 minutes or is about 30 minutes; b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume), optionally wherein the biological sample is diluted to about 1: 10, wherein terminating the treatment is inactivation of the treatment, wherein is inactivation of the treatment is inactivation of the proteinase, wherein inactivation of the treatment includes incubation of the biological sample at an inactivation temperature for a period of time, wherein the inactivation temperature is less than about 65 °C, optionally wherein the inactivation temperature is about 55 °C, wherein the period of time is 5-15 minutes or is about 10 minutes, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40, and wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step; wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, optionally wherein the whole blood sample comprises capillary blood, wherein the plasma sample volume comprises 1-100 pL, and the blood sample volume comprises 2.5-500 pL, optionally wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL, wherein the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is associated with a phenotypic trait, a congenital disease, or a genetic disorder; the method further includes detecting a congenital disease or genetic disorder or determining a phenotypic trait of the fetus wherein: i) the -32- ACTIVE\1627041887.1presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the congenital disease, genetic disorder, or phenotypic trait, or ii) the absence of the target nucleic acid is detected and the fetus is determined not to have or likely develop the congenital disease, genetic disorder, or phenotj pic trait.

[0072] In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a sample from a subject includes detecting the presence or absence of the target nucleic acid in a biological sample from the subject using the method including a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, wherein the biological sample is plasma, wherein the method further includes separating the plasma from a whole blood sample using centrifugation, optionally wherein the whole blood sample comprises capillary blood, wherein the plasma sample volume comprises 1-100 pL, and the blood sample volume comprises 2.5-500 pL, optionally wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL. wherein the treatment is a proteinase, optionally proteinase K, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment, wherein step a) further includes applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is SDS (e.g., 0.005-0.2%, optionally 0.02%), wherein step a) further includes activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a period of time, wherein the activation temperature is greater than 20 °C, optionally wherein the activation temperature is about 37 °C, and wherein the period of time is 20-40 minutes or is about 30 minutes; b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume), optionally wherein the biological sample is diluted to about 1:10, wherein terminating the treatment is inactivation of the treatment, wherein is inactivation of the treatment is inactivation of the proteinase, wherein inactivation of the treatment includes incubation of the biological sample at an inactivation temperature for a period of time, wherein the inactivation temperature is less than about 65 °C, optionally wherein the inactivation temperature is about 55 °C, wherein the period of time is 5-15 minutes or is about 10 minutes, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological -33- ACTIVE\1627041887.1sample, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40, and wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step; wherein the nucleic acid is a tumor cell-derived nucleic acid, and wherein the target nucleic acid is associated with cancer.

[0073] In some embodiments, the method of detecting the presence or absence of a target nucleic acid in a sample from a subject includes detecting the presence or absence of the target nucleic acid in a biological sample from the subject using the method including a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample, wherein the biological sample includes 0.1-1,000 GE of target nucleic acid per mL of biological sample, wherein the biological sample is plasma, wherein the method further includes separating the plasma from a whole blood sample using centrifugation, optionally wherein the whole blood sample comprises capillary blood, wherein the plasma sample volume comprises 1-100 pL, and the blood sample volume comprises 2.5-500 pL, optionally wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL, wherein the treatment is a proteinase, optionally proteinase K, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment, wherein step a) further includes applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is SDS (e.g., 0.005-0.2%, optionally 0.02%), wherein step a) further includes activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a period of time, wherein the activation temperature is greater than 20 °C, optionally wherein the activation temperature is about 37 °C, and wherein the period of time is 20-40 minutes or is about 30 minutes; b) terminating the treatment to the biological sample, wherein upon completion of step b). the biological sample is diluted not more than 1:40 (sample: total volume), optionally wherein the biological sample is diluted to about 1:10, wherein terminating the treatment is inactivation of the treatment, wherein is inactivation of the treatment is inactivation of the proteinase, wherein inactivation of the treatment includes incubation of the biological sample at an inactivation temperature for a period of time, wherein the inactivation temperature is less than about 65 °C, optionally wherein the inactivation temperature is about 55 °C, wherein the -34- ACTIVE\1627041887.1period of time is 5-15 minutes or is about 10 minutes, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b); and c) performing a quantitative polymerase chain reaction (qPCR) using all or an aliquot of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample, wherein the biological sample on which the qPCR is performed is diluted not more than 1:40, and wherein the method is an extraction-free method, and target nucleic acid of the biological sample is not extracted, isolated, and / or purified from the biological sample, for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to the detection step; wherein the nucleic acid is a tumor cell-derived nucleic acid, and wherein the target nucleic acid is associated with cancer; the method further includes detecting minimal residual disease (MRD), wherein: i) the presence of the target nucleic acid is detected and the subject is determined to have MRD, or ii) the absence of the target nucleic acid is detected and the subject is determined to not have MRD.

[0074] Methods, compositions, and devices for fetal sex determination and nucleic acid preservation are described in PCT / US2020 / 052187, PCT / US2021 / 063385, and PCT / US2022 / 074222, each of which patent application is herein incorporated by reference in its entirety, and for disclosure of methods, compositions, and devices for fetal sex determination and nucleic acid preservation. In some embodiments, methods, compositions, and / or devices for fetal sex determination and nucleic acid preservation described in PCT / US2020 / 052187, PCT / US2021 / 063385, and PCT / US2022 / 074222 are utilized in the “extraction-free’' methods disclosed herein.EXAMPLESExample 1 - Extraction-Free Assay for Target Nucleic Acid Detection

[0075] The following assay is utilized in methods to detect the presence or absence of a target nucleic acid in a biological sample, and does not include nucleic acid extraction and / or purification steps.

[0076] 4 pL of 10X Thermolabile Proteinase K (TLPK) was combined with 5 pL of 0.02% SDS per each sample reaction. 9 pL of TLPK / SDS mixture was then added to each well of a clean 0.2 mL semi-skirted PCR plate. A whole blood sample from a patient is centrifuged at 1.6 RCF for 15 minutes. 10 pL of plasma are aliquoted from the centrifuged -35- ACTIVE\1627041887.1sample into each well of the PCR plate containing 9 pL of TLPK / SDS mixture. The PCR plate was then incubated at 37 °C for 30 minutes to activate proteinase K. Following the proteinase K activation step, the PCR plate is then incubated at 55 °C for 10 minutes to deactivate proteinase K. Following the deactivation of proteinase K step, the PCR plate was removed from the incubator and 81 pL of aqueous solution was added to each well of the PCR plate at ambient / room temperature.

[0077] A master mix for the qPCR reaction was prepared by combining the following volumes per sample in Table 1. 18S ribosomal RNA (RNA18s) is utilized as a control reference gene for the qPCR reactions.Table 1: qPCR Master MixVolume of Reagent0.15 pL master mix0.15 pL 50 pM target nucleic acid forwardprimer0.15 pL 50 pM target nucleic acid reverseprimer0.075 pL 50 pM target nucleic acid probe0.1 pL RNA 18s target forward primer0.1 pL RNA18s target reverse primer0.05 pL RNA18s target nucleic acid probe0.0625 pLIM magnesium chloride0.188 pL nuclease-free water

[0078] 4 pL of prepared master mix solution was added to each well of a clean 0.2 mL semi-skirted PCR plate. 21 pL of the treated and diluted plasma sample was added to each well of the PCR plate containing prepared master mix solution. Optical adhesive film is applied to the top of the PCR plate. The PCR plate was then centrifuged for 30 seconds at 2800 rpm. The PCR plate was then inserted into a StepOnePlus qPCR instrument (ThermoFisher Scientific) to conduct the qPCR reaction. The qPCR reaction conditions were set at 3 minutes at 95 °C for the initial denaturation of DNA, followed by 40 cycles of 5 seconds at 95 °C for denaturation and 20 seconds at 60 °C for annealing. The qPCR data is then reviewed and analyzed to detect the presence or absence of the target nucleic acid.-36- ACTIVE\1627041887.1Example 2: Determination of Fetal Sex using the Exemplary' Extraction-Free Assay, and Comparison of the Exemplary Extraction-Free Assay with a Commercial Assay Including cfDNA Extraction in a Fetal Sex Determination

[0079] Whole capillary blood samples were collected from twenty pregnant human subjects. These samples were used to demonstrate the capability of the extraction-free assay to detect fetal sex from unpurified maternal plasma.

[0080] Blood samples were sent to a clinical lab where 50-3000 pL of each collected blood sample was then centrifuged at 1.6 RCF for 15 minutes to separate plasma from whole blood. 10 pL of each plasma sample was utilized in the exemplary extraction-free assay and 100 pL of each plasma sample was utilized in the commercially-available fetal sex determination assay.

[0081] For the exemplary- extraction-free assay, 4 pL of 10X Thermolabile Proteinase K (TLPK) was combined with 5 pL of 0.02% SDS per each sample reaction. 9 pL of TLPK / SDS mixture was then added to each well of a clean 0.2 mL semi-skirted PCR plate.10 pL of each plasma sample was aliquoted into a well of the PCR plate containing 9 pL of TLPK / SDS mixture. The PCR plate was then incubated at 37 °C for 30 minutes to actiyate proteinase K. Following the proteinase K activation step, the PCR plate was then incubated at 55 °C for 10 minutes to deactivate proteinase K. Following the deactivation of proteinase K step, the PCR plate was removed from the incubator and 81 pL of aqueous solution was added to each well of the PCR plate at ambient / room temperature.

[0082] A master mix for the qPCR reaction was prepared by combining the following volumes per sample in Table 1. The target forward and reverse primers were designed to amplify a target sequence on the Y -chromosome in order to detect the presence or absence of male cell-free fetal DNA. 18S ribosomal RNA (RNA18s) was utilized as a control reference gene for qPCR. 4 pL of prepared master mix solution was added to each well of a clean 0.2 mL semi-skirted PCR plate. 21 pL of the treated and diluted plasma sample was added to each well of the PCR plate containing prepared master mix solution. Optical adhesive film was applied to the top of the PCR plate. The PCR plate was then centrifuged for 30 seconds at 2800 rpm. The PCR plate was then inserted into a StepOnePlus qPCR instrument (ThermoFisher Scientific) to conduct the qPCR reaction. The qPCR cycling conditions were set at 3 minutes at 95 °C for the initial denaturation of DNA, followed by 40 cycles of 5 seconds at 95 °C for denaturation and 20 seconds at 60 °C for annealing. Two replicates of each plasma sample were tested.-37- ACTIVE\1627041887.1

[0083] For the commercially-available fetal sex determination assay which includes a cfDNA extraction step. 100 pL of each plasma sample was incubated with Proteinase K for 20 minutes at 37 °C. Following proteinase K treatment, cfDNA was isolated from the plasma samples using a MagMAX Cell-Free DNA Isolation Kit (ThermoFisher) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction was utilized to detect male cell-free fetal DNA as follow s. Isolated cell-free DNA (10 pL) was dispensed into 96-well plates and reacted with a custom master mix for a final qPCR reaction volume of 25 pL per well. Male cell-free DNA w as detected using a multi -copy target sequence on the Y-Chromosome. An autosomal control gene was detected to confirm that a sufficient amount of total cfDNA (maternal and fetal) was isolated from the sample. Optical adhesive film was applied to the top of the PCR plate. The PCR plate was then centrifuged PCR plate for 30 seconds at 2800 rpm. The PCR plate was then inserted into a StepOnePlus qPCR instrument (ThermoFisher Scientific) to conduct the qPCR reaction. The qPCR cycling conditions were set at 3 minutes at 95 °C for the initial denaturation of DNA, followed by 45 cycles of 15 seconds at 95 °C for denaturation and 30 seconds at 60 °C for annealing.

[0084] The qPCR data from the exemplary extraction-free assay and the commercially-available fetal sex determination assay were then reviewed and analyzed to detect the presence or absence of the male cell-free fetal DNA in each plasma sample. An algorithm that incorporated the quantification cycle (Cq) value of the Y-chromosome target sequence and autosomal control gene qPCR reactions was utilized to determine fetal sex. Table 2 shows the fetal gestational age at the time of sample collection, the Y-chromosome target sequence Cq and Autosomal Control Cq values for each sample from the commercially-available fetal sex determination assay, and the Y-chromosome target sequence Cq and Autosomal Control Cq values for each sample from the exemplary extraction-free assay. The fetal gestational age ranged from 8-13.29 weeks at the time of sample collection. The average Cq value for the Y-chromosome target sequence for plasma from male bearing pregnancies using the commercially-available fetal sex determination assay was 27.41, with a range of 26.58-28.86. The average Cq value for the Y-chromosome target sequence for plasma from male bearing pregnancies from the first replicate of the exemplary extraction-free assay was 29.63, with a range of 28.58-31.29. The average Cq value for the Y-chromosome target sequence for plasma from male bearing pregnancies from the second replicate of the exemplary extraction-free assay was 29.62, with a range of 28.53-31.29.-38- ACTIVE\1627041887.1Table 2: Cq Values from the Exemplary Extraction-Free Assay and Commercially-Available Assay _ _ _Gestation Commercial Gender Commercialassay - assay - Y Chromosome Autosomaltarget Cq Value Ctrl Cqvalue1 8.14 27.72 Boy 32.342 11 26.71 Boy 31.643 9.71 27.5 Boy 31.434 7.71 27.38 Boy 31.85 9.14 26.58 Boy 31.826 13.29 28.86 Boy 337 8 27.99 Boy 31.978 10.14 27.31 Boy 32.089 unknown 27.16 Boy 32.3310 10 26.85 Boy 31.9711 15 Undetermined Girl 32.7412 8.71 Undetermined Girl 32.4313 6.29 Undetermined Girl 32.2114 9.14 Undetermined Girl 32.9415 7.29 Undetermined Girl 31.8116 786 Undetermined Girl 32817 12.57 Undetermined Girl 32.8218 12.71 Undetermined Girl 32.819 9.86 Undetermined Girl 31.0720 11.57 Undetermined Girl 32.9226.98 29.42 Pos45 30.56 Neg3976 4085 NTC-39- ACTIVE\1627041887.1Table 2: Cq Values from the Exemplary Extraction-Free Assay and Commercially-Available Assay (continued)Repeat 1 Repeat 2 Gestation Without plasma Without Without Without purification - Y plasma plasma plasma Chromosome purification - purification - Y purification - target Cq Value Autosomal Ctrl Chromosome Autosomal Cq value target Cq Ctrl Cq value Value1 8.14 30.43 31.89 30.35 32.04 2 11 29.42 32.01 29.18 32.01 3 9.71 29.12 31.1 29.18 31.31 4 7.71 30.59 32.31 30.76 32.45 5 9.14 28.93 31.47 29.04 31.93 6 13.29 31.29 33.53 31.29 33.25 7 8 28.99 31.38 29.26 31.82 8 10.14 29.59 31.53 294 31.56 9 unknown 29.33 32.72 29.24 32.89 10 10 28.58 31.42 28.53 31.55 11 15 Undetermined 32.37 36.13 32.12 12 8.71 36.21 32.5 36.91 32.1 13 6.29 Undetermined 32.52 Undetermined 32.36 14 9.14 Undetermined 32.89 Undetermined 32.26 15 7.29 Undetennined 32 Undetennined 31.34 16 7.86 Undetermined 33.71 Undetermined 33.15 17 12.57 Undetermined 31.75 Undetermined 31.94 18 12.71 Undetennined 32.37 Undetennined 32.69 19 9.86 Undetermined 29.73 36.01 29.72 20 11 57 Undetermined 3043 Undetermined 306 Pos 28.55 27.93Neg 35.96 29.74NTC Undetermined Undetermined

[0085] Total cfDNA for both male bearing pregnancies and female bearing pregnancies were comparable for both assays. The average Cq value for the autosomal control for plasma from male bearing pregnancies using the commercially-available fetal sex determination assay was 32.04, with a range of 31.43-33. The average Cq value for the autosomal control for plasma from female bearing pregnancies using the commercially-available fetal sex determination assay was 32.45, with a range of 31.07-32.94. The average Cq value for the autosomal control target sequence for plasma from male bearing pregnancies from the first replicate of the exemplary extraction-free assay was 31.94, with a range of 31.1 -33.53. The average Cq value for the autosomal control target sequence for plasma from female bearing pregnancies from the first replicate of the exemplary extraction-free assay was 32.03, with a range of 29.73-33.71. The average Cq value for the autosomal control target sequence for plasma from male bearing pregnancies from the second replicate of the exemplary extraction-free assay was 32.08, with a range of 31.31-33.25. The average Cq value for the-40- ACTIVE\1627041887.1autosomal control target sequence for plasma from male bearing pregnancies from the second replicate of the exemplary extraction-free assay was 31.83, with a range of 39.72-33.15.

[0086] FIGs. 1A-B shows Cq value separation based on fetal sex with male samples represented with diamonds and female samples represented by circles. FIG. 1A shows Cq values from plasma samples tested with the commercially-available assay and FIG. 1B shows Cq values from plasma samples tested with the exemplary extraction-free assay.

[0087] FIGs. 2A-B shows qPCR amplification plots of male fetus samples #1, #2, #3, #4 tested with the commercially-available assay (FIG. 2A) and the exemplary extraction-free assay (FIG. 2B). FIGs. 3A-B shows the qPCR application plots of female fetus samples #11, #12, #13 tested with the commercially-available assay (FIG. 3A) and the exemplary extraction-free assay (FIG. 3B).

[0088] Following the qPCR analysis, qPCR products from the exemplary extraction-free assay were purified using a DNA extraction kit. To validate the fetal sex determination, gel electrophoresis was performed using the purified qPCR products on a 4% agarose gel. FIG. 4 shows a picture of the gel electrophoresis with purified qPCR products from the exemplary extraction-free assay. The lane assignments correspond to M: Molecular weight marker; lane 1: Male Sample #1 - Replicate 1, lane 2: Male Sample #2 - Replicate 1, lane 3: Male Sample #3 - Replicate 1, lane 4: Male Sample #4 - Replicate 1, lane 5: Female Sample #11 - Replicate 1, lane 6: Female Sample #12 - Replicate 1, lane 7: Female Sample #13 - Replicate 1. lane 8: negative control, lane 9: positive control, lane 10: non-template control. The gel electrophoresis results confirm the amplification of the target Y-chromosome sequence.

[0089] These results demonstrate that the exemplary extraction-free assay effectively minimizes the required amount of plasma compared to the commercially-available assay which includes a cfDNA extraction step and accurately determined fetal sex in cases where a female was carrying a male fetus.

[0090] Although the foregoing methods have been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modifications and alternatives coming with the true scope and spirit of the embodiments of the disclosure.-41- ACTIVE\1627041887.1

[0091] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like.

[0092] The indefinite article “a” or “an’’ does not exclude a plurality. The use of “about’’ before a number includes the number itself. For example, “about 5’’ provides express support for “5”.-42- ACTIVE\1627041887.1

Claims

WHAT IS CLAIMED IS:

1. A method of detecting the presence or absence of a target nucleic acid in a biological sample, the method comprising:a) applying a treatment to a biological sample to release nucleic acids bound to proteins in the biological sample;b) terminating the treatment to the biological sample, wherein upon completion of step b), the biological sample is diluted not more than 1:40 (sample: total volume); and c) performing a quantitative polymerase chain reaction (qPCR) using all or a portion of the biological sample to detect the presence or absence of the target nucleic acid in the biological sample.

2. The method of claim 1. wherein the biological sample on which the qPCR is performed is diluted not more than 1:40.

3. The method of claim 1 or 2, wherein the treatment is a proteinase, optionally proteinase K.

4. The method of any one of claims 1-3, wherein terminating the treatment is inactivation of the treatment, optionally inactivation of the proteinase.

5. The method of claim 4, wherein inactivation of the treatment comprises incubation of the biological sample at an inactivation temperature for a first period of time.

6. The method of claim 5, wherein the inactivation temperature is less than about 65 °C.

7. The method of claim 5 or 6, wherein the inactivation temperature is about 55 °C or about 45-65 °C, 45-60 °C, 50-65 °C, 50-60 °C, or 52-57 °C.

8. The method of any one of claims 5-7, wherein the first period of time is about 10 minutes or about 5-25 minutes, 5-20 minutes, 10-25 minutes, 5-15 minutes, or 8-12 minutes.

9. The method of any one of claims 1-8, wherein the method further comprises activation of the treatment after it is applied to the biological sample by incubation of the biological sample at an activation temperature for a second period of time.

10. The method of claim 9. wherein the activation temperature is greater than 20 °C.

11. The method of claim 9 or 10. wherein the activation temperature is about 37 °C, or about 20-42 °C, 27-42 °C, 27-40 °C, 30-42 °C, 30-40 °C, or 35-40 °C.

12. The method of any one of claims 9-11, wherein the second period of time is about 30 minutes, or about 10-50 minutes, 10-40 minutes, 20-50 minutes, 20-40 minutes, or 25-35 minutes.-43- ACTIVE\1627041887.

113. The method of any one of claims 1-12, wherein the biological sample is incubated at ambient / room temperature during the application of the treatment in step a).

14. The method of any one of claims 1-13, wherein the biological sample is incubated at ambient / room temperature after the termination of the treatment in step b).

15. The method of any one of claims 1-14, wherein upon completion of step b) or wherein the biological sample on which the qPCR is performed, the biological sample is diluted to about 1:1.25, 1:1.5, 1:

2. 1:4, 1:5, 1:6, 1:

7. 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, or a range defined by any two of the preceding values, optionally 1: 1.25 to 1:35, 1:2 to 1:20, or 1:5 to 1:10.

16. The method of any one of claims 1-15, wherein the biological sample is diluted to about 1:10.

17. The method of any one of claims 1-16, wherein:wherein said applying a treatment to a biological sample comprises treatment with proteinase K and incubating the biological sample at about 37 °C for about 30 minutes;wherein said terminating the treatment to the biological sample comprises incubating the biological sample at about 55°C for about 10 minutes; and wherein upon completion of step b) the biological sample is diluted to about 1:10.

18. The method of any one of claims 1-17, wherein step a) further comprises applying at least one chemical reagent in addition to the treatment to the biological sample, optionally wherein the at least one chemical reagent is a detergent, optionally wherein the detergent is sodium dodecyl sulfate (SDS), optionally wherein the detergent is applied to the biological sample at a concentration that is, is about, is not more than, is not less than, 0.001, 0.002, 0.005, 0.01, 0.

02. 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 12, 15, or 20%, or a range defined by any two of the preceding values, for example, 0.001-20, 0.001-1. 0.002-2, 0.05-10. 0.1-10, or 0.002-0.5%, optionally 0.005-0.2%, or 0.02%.

19. The method of any one of claims 1-18, wherein dual-labeled probes are utilized in the qPCR of step c, optionally wherein the dual-labeled probes each comprise a minor groove-binding moiety.

20. The method of any one of claims 1-19. wherein the biological sample volume is less than about 500 pL, optionally wherein the biological sample volume is less than about 100 pL.

21. The method of any one of claims 1-20, wherein the biological sample volume is about 10 pL or about 1-500 pL, 2-500 pL, 5-500 pL, 10-500 pL, 100-400 pL, 1-100 pL. 5-50 pL, 50-100 pL, 5-20 pL, 5-15 pL, or about 1-12 pL.-44- ACTIVE\1627041887.

122. The method of any one of claims 1-21, wherein the biological sample is blood, plasma, serum, saliva, urine, and / or cervical mucus, optionally wherein the biological sample is treated with a preservative.

23. The method of any one of claims 1-21, wherein the biological sample is plasma.

24. The method of claim 23, further comprising separating the plasma from a whole blood sample using centrifugation, optionally wherein the whole blood sample comprises capillary blood.

25. The method of claim 24, wherein the plasma sample volume is about 10 pL, and the whole blood sample volume is about 25 pL, optionally wherein the plasma sample volume is about 2 pL, and the whole blood sample volume is about 5 pL.

26. The method of claim 24, wherein the plasma sample volume comprises about 1-100 pL, and the blood sample volume comprises about 2.5-500 pL.

27. The method of any one of claims 1-26, wherein the target nucleic acid is DNA or RNA.

28. The method of any one of claims 1 -27, wherein biological sample is from a pregnant subject and the target nucleic acid is a fetal nucleic acid.

29. The method of any one of claims 1-27, wherein the sample is from a subject, and the target nucleic acid is a genomic nucleic acid of the subject, optionally wherein the target nucleic acid is a tumor cell-derived nucleic acid.

30. The method of any one of claims 1-29. wherein the target nucleic acid is a cell-free nucleic acid, optionally wherein the target cell-free nucleic acid is a cell-free DNA (cfDNA).

31. The method of claim 30, wherein the biological sample comprises a concentration of total cfDNA per sample volume (ng / mL), optionally wherein the concentration of total cfDNA per sample volume is 0.0003, 0.0006, 0.001, 0.003, 0.

006. 0.

01. 0.

03. 0.

06. 0.1, 0.2, 0.3, 0.

4. 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any tw o of the preceding values, for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, 0.01-3 ng / mL, or 0.03-2 ng / mL, optionally 0.5 ng / mL.

32. The method of claim 31. wherein the target nucleic acid comprises 2-10% of the total abundance / amount of cfDNA in the biological sample.

33. The method of any one of claims 1-32, wherein the biological sample comprises a concentration of target nucleic acid per sample volume (ng / mL), optionally wherein the concentration of target nucleic acid per sample volume is 0.0003, 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.

4. 0.

5. 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.

2. 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or a range defined by any two of the preceding values,-45- ACTIVE\1627041887.1for example 0.0003-10, 0.0003-2, 1-10, 0.0003-0.03, 0.003-3, 0.01-3 ng / mL or 0.03-2 ng / mL, optionally 0.5 ng / mL.

34. The method of any one of claims 1-33, wherein the biological sample comprises 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, or 15000 copies of the target nucleic acid per sample volume (copies / pL) or a range defined by any two of the preceding values, for example 0.1-15000, 1-5000. 1-1000, 5-10000, 5000-15000, 10-5000, 1-200, or 1-50 copies / pL, optionally 15-3000 copies / pL or about 300 copies / pL.

35. The method of any one of the preceding claims, wherein the concentration of the target nucleic acid in the biological sample is, or is less than, 3000, 2500, 2000, 1500, 1000, 500, 250, 100, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 GE / rnL of biological sample, or a range defined by any two of the preceding values, for example 0.1-3000, 500-2500, 1-3000, 0.1-500, 0.1-100, 0.1-50, 1-10, 1-5, 0.5-10, 0.5-5 GE / ml of biological sample, optionally 10-500 GE of target nucleic acid per rnL of sample.

36. The method of any one of claims 1-35, wherein the target nucleic acid is associated with a phenotypic trait.

37. The method of any one of claims 1-36. wherein the target nucleic acid is on the Y-chromosome.

38. The method of any one of claims 1-37, wherein the target nucleic acid is associated with a pathological condition, optionally wherein the pathological condition is a congenital disease or cancer.

39. A method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of claims 1-37,wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, optionally wherein the whole blood sample is a capillary sample, wherein the target nucleic acid is a fetal cell-free nucleic acid, andwherein the target nucleic acid is on the Y chromosome.

40. The method of claim 39, the method further comprising determining the sex of the fetus, wherein:i) the presence of the target nucleic acid is detected and the fetus is determined to be male, orii) the absence of a target nucleic acid is detected and the fetus is determined to be female.-46- ACTIVE\1627041887.

141. A method of determining the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of claims 1-38,wherein the biological sample is plasma separated from a whole blood sample from the pregnant subject, the target nucleic acid is a fetal cell-free nucleic acid, and wherein the target nucleic acid is associated with a phenotypic trait / characteristic 42. The method of claim 41, the method further comprising determining a phenotypic trait of the fetus, wherein:i) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the phenotypic trait, orii) the absence of a target nucleic acid is detected and the fetus is determined not to have or likely develop the phenotypic trait.

43. A method of detecting the presence or absence of a target nucleic acid of a fetus of a pregnant subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the pregnant subject using the method of any one of claims 1-38,wherein the biological sample is plasma separated from a maternal whole blood sample,wherein the nucleic acid is a fetal cell-free nucleic acid, andwherein the target nucleic acid is associated with a congenital disease.

44. The method of claim 43, wherein the method further comprising detecting congenital disease, whereini) the presence of the target nucleic acid is detected and the fetus is determined to have or likely develop the congenital disease, orii) the absence of the target nucleic acid is detected and the fetus is determined not to have or likely develop the congenital disease.

45. A method of detecting the presence or absence of a target nucleic acid in sample from a subject, comprising:detecting the presence or absence of the target nucleic acid in a biological sample from the subject using the method of any one of claims 1-38,wherein the nucleic acid is a tumor cell-derived nucleic acid, andwherein the target nucleic acid is associated with cancer.-47- ACTIVE\1627041887.

146. The method of claim 45, the method further comprising detecting minimal residual disease (MRD), wherein:i) the presence of the target nucleic acid is detected and the subject is determined to have MRD, orii) the absence of the target nucleic acid is detected and the subject is determined to not have MRD.

47. The method of any one of claims 1 -46, wherein the sample is from a human subject.

48. The method of any one of claims 1-47, wherein the method is an extraction-free method.

49. The method of any one of claims 1-48, wherein the method does not comprise extracting, isolating and / or purifying the target nucleic acid from the sample prior to step c), optionally for example by using magnetic bead-based nucleic acid separation technology, phenol-chloroform extraction, alcohol precipitation, Chelex® resin extraction, spin-column based nucleic acid extraction, and / or silica-based nucleic acid isolation prior to step c).-48- ACTIVE\1627041887.1

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