Y chromosome STR gene locus detection kit and application thereof

Abstract

The invention provides a Y chromosome STR gene locus detection kit and application thereof, and belongs to the field of molecular biology. According to the kit, 20 necessary Y chromosome STR sites are amplified, so that efficient and accurate gene detection is realized. The kit comprises a first-step PCR (Polymerase Chain Reaction) primer containing a specific primer and a universal primer for second-step PCR amplification. The 5'end of the first-step PCR primer contains a base sequence which is the same as partial or all sequences of the universal primer, and library preparation can be realized through two-step PCR. Wherein the 5'end of one universal primer is subjected to phosphorylation modification, and the prepared library can be compatible with Illumina and MGI sequencing platforms. The kit is suitable for a complete or highly-degraded genome DNA sample, and the effectiveness and reliability of the kit in various sample types are ensured. The invention provides an innovative solution for Y chromosome STR detection, has the characteristics of high throughput and high compatibility, and can meet the requirements under various scientific research and application scenes.

Description

Technical fields: This invention provides a Y chromosome STR locus detection kit and its application, belonging to the field of molecular biology. Background technology: Short tandem repeats (STRs) are regions in the genome consisting of short, repetitive DNA sequence units, typically each containing 2 to 6 base pairs. The number of repeats varies between individuals, making STRs highly polymorphic. Y-chromosome STRs refer to specific STR loci located on the Y chromosome. Because the Y chromosome exists only in males and is passed from father to son, Y-STRs exhibit familial linkage and paternal inheritance characteristics.

[0001] The polymorphism of Y-STRs makes them important in forensic medicine, criminal investigation, and archaeology. In forensic medicine, Y-STRs are used to analyze paternal relationships and distinguish between multiple male samples, verifying father-son or brother relationships and identifying the DNA contributions of different men in multiple male samples, which is crucial for solving cases. In criminal investigation, Y-STRs aid in the detection of male characteristics and the tracing of paternal genetic information, especially in accurately locating male samples in the presence of female DNA and linking samples from the same paternal lineage in different cases.

[0002] In archaeology, Y-STRs are used in paternal lineage and regional historical studies. By analyzing the Y-STRs of ancient male remains, we can study the paternal lineage of historical groups, explore the migration and evolutionary history of ethnic groups, and reveal the male genetic characteristics and population migration routes of groups in different regions and cultural backgrounds.

[0003] Due to its unique genetic transmission and sex specificity, the Y chromosome STR has become an important tool in forensic science, criminal investigation, and archaeological research, providing a reliable method for analyzing and tracing paternal lineage.

[0004] Currently, the main method for detecting STRs is PCR combined with capillary electrophoresis. The principle is to design a pair of PCR primers on the flanking sequences of the STR locus, label one of the primers with fluorescence, and then analyze the PCR products by capillary electrophoresis to determine the genotype of the STR based on the peak shape. This method has the advantages of high sensitivity and low detection cost. However, there are the following limitations: (1) Limited detection throughput: When designing primers, it is necessary to first understand the repeat length of each STR and carefully adjust the amplicon length to avoid peak overlap of different STRs. When the number of STRs to be detected is large (e.g., more than 40), the complexity of primer design increases significantly. Although different fluorescent labels can be used to distinguish amplicons of similar length, the design is still challenging. In addition, when trying to add STR loci to the existing detection scheme, it is often impossible to achieve due to the overlap of amplicon lengths. (2) Sample condition limitations: In forensic identification, criminal investigation or archaeological sampling, the samples are usually small amounts of hair, skin flakes, blood spots, semen spots, or ancient bone fossils, etc. The genomic DNA content of these samples is extremely low and often highly degraded. For amplicones longer than 200 bp, the amplification efficiency will be significantly reduced, which may lead to decreased detection sensitivity or even false negative results.

[0005] Next-generation sequencing (NGS) is an advanced DNA sequencing technology capable of high-throughput sequencing of large amounts of DNA fragments in a short time. Compared to traditional Sanger sequencing, NGS significantly improves sequencing speed and data output while reducing the sequencing cost per base. Amplicon sequencing is a targeted sequencing method that uses specific primers to perform multiplex PCR amplification of a target gene, followed by sequencing of the amplified products. Compared to probe-based capture sequencing and whole-genome sequencing, amplicon sequencing offers advantages such as shorter detection time, lower cost, and simpler data analysis.

[0006] In STR detection, amplicon sequencing demonstrates significant advantages over multiplex PCR combined with capillary electrophoresis: (1) Flexible primer design: It eliminates the need to distinguish between amplicon lengths of different STRs, increasing the throughput of STR loci detection; (2) High adaptability: It can limit amplicon length to within 200 bp, or even shorter, making it compatible with highly degraded DNA samples and improving the detection capability for low-quality samples; (3) High sample throughput: It can process dozens to hundreds of samples at a time, meeting the needs of large-scale detection. This method significantly improves the efficiency and accuracy of STR detection and is suitable for genetic analysis of various complex samples.

[0007] Therefore, this invention provides a Y-chromosome STR locus detection kit and its application, which can simultaneously detect 20 Y-chromosome STR loci. This technology belongs to the field of molecular biology and is widely used in forensic genetics, archaeology, and other fields. Summary of the Invention: This invention relates to a gene detection kit for detecting Y chromosome short tandem repeats (STRs). Through a series of innovative designs, this kit significantly improves the detection sensitivity and specificity of STR loci, making it particularly suitable for the rapid and efficient detection of Y chromosome STRs in DNA samples with highly degraded DNA or low template content.

[0008] The kit includes amplification reagents for 20 Y chromosome STR loci, including first-step PCR primers and universal primers for second-step PCR amplification. The first-step PCR primers consist of a specific primer sequence and a common tag sequence. The specific primer precisely matches the flanking sequences of the target locus, while the common tag sequence has the same or similar base sequence as the universal primer. This design ensures specific PCR amplification and efficient amplification of the target region.

[0009] Furthermore, the common tag sequence is compatible with various Illumina library adapter sequences, including Nextera, Ampliseq, and TruSeq adapter sequences. This compatibility design allows the prepared libraries to be adapted to multiple sequencing platforms, greatly expanding the application range and market potential of the kit. In addition, to enhance library stability and compatibility with sequencing platforms, the 5' end of one of the universal primers is phosphorylated, allowing the library to be directly circularized for compatibility with the MGI sequencing platform.

[0010] This invention is specifically implemented through the following steps: 1. Multiple primer design and screening: This invention selects 20 essential STR loci from 60 recommended by public security authorities to design and screen optimal primer combinations. Through precise primer design and optimization strategies, it ensures effective amplification of genomic DNA, including both intact and degraded DNA. This invention integrates resources from multiple databases, including NCBI, YHRD, and UCSC, to obtain the chromosomal coordinates of the STR loci and designs specific primers for each STR. Primer design follows these principles: optimizing amplicon length, the Tm value range for specific binding, and avoiding the formation of poly(N) structures. Optimal primer combinations (SEQ ID NO. 7 to SEQ ID NO. 42) are selected by evaluating primer cross-dimer interactions.

[0011] 2. Amplicon library preparation: A two-step PCR amplification strategy was employed. The first step used first-step PCR primers containing a common tag sequence and specific primer sequences to identify flanking sequences of the STR locus. The second step used partial or complete sequences of Illumina adapter sequences as universal primers to integrate the complete adapter into the target amplification sequence. This strategy effectively improved primer amplification efficiency while reducing non-specific interactions. Specifically, the first base at the 5' end of one universal primer sequence was phosphorylated to meet the library circularization requirements before MGI. The common tag sequences are SEQ ID NO.1 to SEQ ID NO.6.

[0012] 3. Library quality control and sequencing: During library construction, AMPure XP magnetic beads were used for purification, and precise quantification and distribution detection were performed using the Qubit dsDNA HS AssayKit and Agilent 4200 TapeStation system. Evaluation on Illumina and MGI sequencing platforms showed that this method has good compatibility with DNA from different sources (including intact and degraded samples) and exhibits high efficiency and consistency.

[0013] In summary, this invention ensures efficient library construction while meeting the stringent requirements of sequencing platforms for sample diversity compatibility, providing an innovative and efficient solution for forensic, criminal investigation, and human population DNA polymorphism research.

[0014] Advantages of this invention: (1) Strong sample compatibility: suitable for samples with low DNA content and high degradation; (2) High throughput: capable of simultaneously detecting dozens to hundreds of STRs; (3) Good scalability: It is easy to add new detection targets to the existing STR detection panel; (4) Strong platform compatibility: It is applicable to Illumina and MGI sequencing platforms. Attached image description: Figure 1 Illumina library preparation process Detailed implementation method: The invention will now be described in detail with reference to specific embodiments.

[0015] Example 1: Multiple Primer Design Based on the recommendations of the public security authorities, we designed primers for 20 essential STR loci.

[0016] 1. Primer design Chromosomal coordinates for each STR locus were obtained from NCBI and the Y-STR Haplotype Reference Database (YHRD). These coordinates were then extended upstream and downstream by 400 bp from the UCSC database to download the corresponding gene sequences. Next, primers were designed using Primer 3.0 software. The following principles were followed when designing primers: 1) Amplicon length should be between 80 and 200 bp, with an optimal length of 80 to 150 bp; 2) Primer Tm values ​​should be between 55 and 61℃, with an optimal Tm of 59 to 61℃; 3) Poly(N) structures should be avoided; 4) 1 to 3 primer pairs should be designed for each STR locus. Furthermore, primers for STRs located close to each other on chromosomes can be combined. Table 1 lists the primers designed for the 20 STR loci.

[0017] Table 1 Primer sequences for 20 STR loci Note: Due to the close proximity of the sites, primers for DYS385a and DYS385b, and DYS389I and DYS389II were designed in combination (DYS385a / b and DYS389I / II).

[0018] 2. Primer screening Based on the cross-dimer interactions between primers, the combination with the fewest interactions is selected as the optimal combination.

[0019] When constructing libraries for the Illumina sequencing platform, a partial i5 adapter sequence is added to the 5' end of the preferred forward primer, and a partial i7 adapter sequence is added to the 5' end of the preferred reverse primer, serving as their common tag sequences. This common tag sequence is partially complementary to the full-length i5 and i7 adapter sequences, thus adding adapter sequences for cluster generation during primer amplification. This process ensures reduced non-specific interactions during primer binding while facilitating efficient sequencing. Table 2 details the common tag sequences that can be added to the 5' end of the preferred primers.

[0020] The preferred primers for adding adapter sequences to the Ampliseq library are detailed in Table 3.

[0021] Table 2. Common tag sequences at the 5' end of preferred forward and reverse primers. Table 3. Preferred primer combinations (with added Illumina AmpliSeq adapter sequences) Note: The italicized text is a common tag sequence.

[0022] Example 2: Library Preparation To evaluate the amplification effect of the selected primers on intact genomic DNA and degraded genomic DNA, we extracted genomic DNA from oral swab samples and dried blood smears stored at room temperature for one year, constructed libraries, and conducted comparative tests.

[0023] 1. Genomic DNA extraction Exfoliated cells were collected from the oral cavity lining using swabs. Genomic DNA was extracted from 10 oral swab samples using an oral swab genomic DNA extraction kit (TIANGEN, catalog number: DP322). Simultaneously, 10 dried blood smear samples stored at room temperature for one year were collected. Three 3×3 mm dried blood smears from each sample were placed in 1.5 mL centrifuge tubes, and genomic DNA was extracted using a dried blood spot genomic DNA extraction kit (TIANGEN, catalog number: DP334). The concentration and fragment size of the extracted oral swab genomic DNA and dried blood spot DNA were determined using a Qubit dsDNA HS Assay Kit and agarose gel electrophoresis.

[0024] 2. Library preparation Library preparation is achieved through a two-step PCR process. In the first PCR step, specific primers carrying the common tag sequence specifically bind to the flanking sequences of the STR site, thereby amplifying the target sequence. In the second PCR step, Illumina adapter sequences are used as universal primers to amplify the sequencing adapters and ligate them to both ends of the target fragment.

[0025] (1) Primer ratio optimization The primer powders in Table 3 were dissolved in nuclease-free water to prepare a 100 μM stock solution. Then, the primers were mixed at equal concentrations to prepare a working solution with a final concentration of 0.1 μM. Library preparation was performed using genomic DNA from oral swabs, followed by analysis of the sequencing data. The primer concentration ratios were adjusted according to the coverage depth of each STR to ensure uniform coverage for all STRs.

[0026] (2) First step PCR Prepare the amplification mixture on ice according to the following system, and include a negative control (without added genomic DNA) for contamination control. Perform PCR amplification according to Table 5. PCR amplification was performed using a high-fidelity DNA polymerase (TIANGEN, catalog number: EP101). The starting amount of genomic DNA from both oral swabs and dried blood smears was the same.

[0027] Table 4. First step PCR reaction system Table 5. Step 1 PCR reaction procedure (3) Product purification PCR products were purified using Agencourt AMPure XP (Beckman Coulter). AMPure XP beads were equilibrated at room temperature for 30 minutes and then vortexed to mix thoroughly.

[0028] (a) Add 40 μL of well-mixed AMPure XP magnetic beads to the PCR product and mix thoroughly using a vortex mixer; (b) Incubate at room temperature for 15 minutes; (c) Briefly centrifuge and place the PCR tube on a magnetic rack at room temperature for 5 minutes; (d) Remove the supernatant with a pipette, being careful not to pick up the magnetic bead; (e) Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 30 seconds, and remove the ethanol with a pipette; (f) Repeat step (e), briefly centrifuge the PCR tube and place it on a magnetic rack, then use a pipette to remove any remaining 80% ethanol; (g) Open the PCR tube cap and allow it to dry at room temperature for 1 minute; (h) Add 30 μL of 10 mM Tris-HCl (pH 8.5) to the PCR tube, mix well with a vortex mixer, and incubate at room temperature for 5 minutes; (i) After briefly centrifuging the PCR tube, place it on a magnetic rack and let it stand for 5 minutes; (j) Transfer the supernatant to a new PCR tube using a pipette.

[0029] (4) Second step PCR The purified first-step PCR product was amplified using adapters from the Illumina sequencing platform as universal primers. Specific adapter sequences are detailed in Table 6. One adapter's 5' end first base was phosphorylated to adapt for library circularization used in MGI sequencing. The adapter sequence contains a 6-8 base index, which can be used for sample splitting of the sequencing data. In this invention, the second-step PCR amplification used AmpliSeq adapter sequences as universal primers.

[0030] Table 6. Connector sequences of different libraries Note: i5 and i7 are indexes of 6 to 8 bases.

[0031] Prepare the amplification mixture on ice according to Table 7, and perform PCR amplification according to Table 8. PCR amplification was performed using a high-fidelity DNA polymerase (TIANGEN, catalog number: EP101).

[0032] Table 7. Second-step PCR reaction system Table 8. Second-step PCR reaction procedure (5) Product purification The purification steps are the same as in step (3).

[0033] (6) Document quality inspection Take 1 μL of purified PCR product and perform quantitative analysis using the Qubit dsDNA HS Assay Kit; take another 2 μL of purified PCR product and detect the distribution of library fragments using the DNA1000 high-sensitivity chip on the Agilent 4200 TapeStation system.

[0034] 3. Sequencing (1) On-machine setup with Illumina sequencer Based on the Qubit concentration of each library, the libraries were mixed at equimolar concentrations and then sequenced using a sequencing machine with a read length of PE150.

[0035] (2) On the MGI sequencer The library was circularized and loaded onto the sequencing machine according to the MGI sequencing platform operation guide.

[0036] 4. Sequencing results The genomic DNA from dried blood smears showed poor integrity and severe degradation, while the genomic DNA from oral swabs exhibited good integrity. However, the library concentrations prepared from the same starting amount of genomic DNA were comparable, with no significant difference (Table 9), indicating that the selected primer combinations and library preparation reagents had good compatibility with degraded genomic DNA. Table 10 shows that the library fragment distribution of genomic DNA from oral swabs and dried blood smears was not significantly different between the two samples, and the peak values ​​in the peak plots were similar. Analysis of the processed data showed that the average coverage depth of STR loci was comparable (612X from oral swabs vs. 605X from dried blood smears), and the STR detection results were consistent.

[0037] Table 9. Library concentrations for genomic DNA preparation from oral swabs and dried blood smears. Table 10. Fragment distribution of genomic DNA libraries from oral swabs and dried blood smears. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A Y-chromosome STR gene detection kit, characterized in that, This kit contains first-step PCR primers for amplifying 20 Y chromosome STR loci and universal primers for second-step PCR amplification.

2. The reagent kit according to claim 1, characterized in that, The first step of PCR primers consists of specific primer sequences and a common tag sequence that is partially or completely identical to the universal primer sequence.

3. The reagent kit according to claim 2, characterized in that, The common tag sequences are part and all of the Illumina library connector sequences.

4. The reagent kit according to claim 2, characterized in that, The common tag sequence is part or all of the Illumina Nextera library adapter sequence, including: The common tag sequence located at the 5' end of the forward primer of the first step PCR primer is SEQ ID NO.1; The common tag sequence located at the 5' end of the reverse primer of the first step PCR primer is SEQ ID NO.

2.

5. The reagent kit according to claim 2, characterized in that, The common tag sequence is part or all of the Illumina Ampliseq library adapter sequence, including: The common tag sequence located at the 5' end of the forward primer of the first step PCR primer is SEQ ID NO.3; The common tag sequence located at the 5' end of the reverse primer of the first step PCR primer is SEQ ID NO.

4.

6. The reagent kit according to claim 2, characterized in that, The common tag sequence is part or all of the Illumina TruSeq library adapter sequence, including: The common tag sequence located at the 5' end of the forward primer of the first step PCR primer is SEQ ID NO.5; The common tag sequence located at the 5' end of the reverse primer of the first step PCR primer is SEQ ID NO.

6.

7. The kit according to claim 1, characterized in that, The first step involves using PCR primers complementary to the flanking sequences of 20 Y chromosome STR loci to amplify DNA fragments containing STR sequences, including: The forward and reverse primer sequences for DYS19 amplification are SEQ ID NO.7 and SEQ ID NO.8; The forward and reverse primer sequences for DYS385a / b amplification are SEQ ID NO. 9 and SEQ ID NO. 10; The forward and reverse primer sequences for DYS389I / II amplification are SEQ ID NO.11 and SEQ ID NO.12; The forward and reverse primer sequences used for DYS390 amplification are SEQ ID NO.13 and SEQ ID NO.14; The forward and reverse primer sequences used for DYS391 amplification are SEQ ID NO.15 and SEQ ID NO.16; The forward and reverse primer sequences used for DYS392 amplification are SEQ ID NO.17 and SEQ ID NO.18; The forward and reverse primer sequences used for DYS393 amplification are SEQ ID NO.19 and SEQ ID NO.20; The forward and reverse primer sequences for DYS437 amplification are SEQ ID NO.21 and SEQ ID NO.22; The forward and reverse primer sequences for DYS438 amplification are SEQ ID NO.23 and SEQ ID NO.24; The forward and reverse primer sequences for DYS439 amplification are SEQ ID NO.25 and SEQ ID NO.26; The forward and reverse primer sequences for DYS456 amplification are SEQ ID NO.27 and SEQ ID NO.28; The forward and reverse primer sequences for DYS458 amplification are SEQ ID NO.29 and SEQ ID NO.30; The forward and reverse primer sequences used for DYS448 amplification are SEQ ID NO.31 and SEQ ID NO.32; The forward and reverse primer sequences for DYS635 amplification are SEQ ID NO.33 and SEQ ID NO.34; The forward and reverse primer sequences for YGATAH4 amplification are SEQ ID NO.35 and SEQ ID NO.36; The forward and reverse primer sequences for DYS481 amplification are SEQ ID NO.37 and SEQ ID NO.38; The forward and reverse primer sequences used for DYS533 amplification are SEQ ID NO.39 and SEQ ID NO.40; The forward and reverse primer sequences used for DYS576 amplification are SEQ ID NO.41 and SEQ ID NO.

42.

8. The reagent kit according to claim 1, characterized in that, One of the universal primers used for the second step of PCR amplification has a phosphorylated first base at the 5' end.

9. The application of the kit according to claim 1 in detecting Y chromosome STRs in DNA samples, characterized in that, Suitable for Y chromosome STR detection of DNA with high degradation or low template content.

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