MiniSTR fluorescent multiplex amplification system and kit with 18 short tandem repeat sequences

Abstract

This invention relates to the field of biotechnology, specifically to a MiniSTR fluorescent multiplex amplification system and kit containing 18 short tandem repeat sequences. It discloses primer sequences targeting 17 autosomal STR loci and 1 sex locus. The core PCR products of these specific amplification primers are all less than 300 bp, increasing the number of loci detected in degraded samples. This system offers advantages such as speed, high sensitivity, and adaptability for detecting trace amounts or degraded samples, making it suitable for highly degraded or inhibitory samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a MiniSTR fluorescent multiplex amplification system and kit containing 18 short tandem repeat sequences. It discloses primer sequences targeting these 18 sites, with the core PCR products of the specific amplification primers all being less than 300 bp. This increases the number of loci detected in degraded samples. For the detection of trace amounts, degradation, and other difficult samples, it offers advantages such as speed, high sensitivity, and strong adaptability, making it suitable for highly degraded or inhibitory samples. Background Technology

[0002] Short tandem repeat locus (STR), also known as microsatellite DNA or simple sequence repeats (SSR), are tandem repeats of 2-7 bases as a core sequence found in genes and extragenic regions of the human genome. The number of tandem repeats is approximately 10-60, and the length is generally less than 400 bp. Variations exist between individuals, and they follow Mendelian inheritance, exhibiting dominant inheritance among parents. Due to differences in base composition and length polymorphism, STRs are genetically polymorphic. Forensic molecular biology examination utilizes STR multiplex amplification detection technology as a primary means of evidence examination. It offers significant advantages in terms of sample requirements, sensitivity, and individual identification rate, thus finding widespread application in individual identification and paternity testing.

[0003] In actual cases, crime scenes are often complex, and human tissue is susceptible to damage from adverse environmental conditions such as soil microbial decomposition, treatment with acidic formalin fixatives, high-temperature cauterization, and seawater immersion. These conditions are detrimental to sample preservation, causing DNA double-strand breaks, irreversible denaturation, and making sampling difficult. Therefore, forensic samples are often in trace or ultra-trace quantities, exhibiting changes in DNA molecular structure, reduced DNA content, and increased inhibitor concentrations. Furthermore, samples may experience fragment loss due to degradation. In such cases, traditional STR testing may result in "dominant amplification" or "invalid amplification," leading to the loss of alleles or loci, making it difficult to obtain complete and clear typing and posing significant challenges to individual identification. Therefore, how to conduct DNA testing on degraded and trace biological samples has become a major challenge in forensic biological evidence examination. Summary of the Invention

[0004] The purpose of this invention is to provide a MiniSTR fluorescent multiplex amplification system and kit for detecting 18 short tandem repeat sequences in trace amounts and degraded DNA samples, which is designed for difficult cases in the forensic examination process and features six-color fluorescent labeling.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0006] Based on the loci and arrangement included in ABI's VeriFiler Plus PCR amplification kit and YueWei's autosomal 30A STR kit, 17 autosomal STR loci and 1 sex locus were selected, including CSF1PO, D6S477, D19S253, D8S1132, D5S818, D3S1358, D15S659, D6S1043, D10S1435, Penta E, TPOX, D3S3045, FGA, D7S820, D10S1248, D2S1338, Penta D, and the sex locus Amelogenin. A kit capable of simultaneously amplifying these 17 autosomal STR loci and 1 sex locus was developed.

[0007] The primers and their corresponding concentrations are as follows (Table 1):

[0008] Table 1: Primer sequences and concentration ratios for each locus in the multiplex amplification system

[0009]

[0010] The amplification sites in the amplification kit are labeled with five different colors of fluorescent markers. The same fluorescent marker is considered as one group. The five groups are as follows:

[0011] Group 1: CSF1PO, D6S477, D19S253, D8S1132;

[0012] Group 2: Amelogenin, D5S818, D3S1358, D15S659;

[0013] Group 3: D6S1043, D10S1435, Penta E;

[0014] Group 4: TPOX, D3S3045, FGA, D7S820;

[0015] Group 5: D10S1248, D2S1338, Penta D.

[0016] The internal standard used in this kit is labeled with the orange fluorescent marker ORG, which is the sixth group of fluorescent markers.

[0017] The six groups of fluorescent labels were FAM, HEX, TAMRA, ROX, PURP, and ORG.

[0018] The first group is labeled with FAM, the second with HEX, the third with TAMRA, the fourth with ROX, and the fifth with PURP. The internal standard used for detection in this multiplex amplification system is labeled with the orange fluorophore ORG, which is the sixth fluorescent label.

[0019] The present invention also provides a multiplex amplification kit, comprising the above-described multiplex amplification system.

[0020] The multiplex amplification kit also includes PCR Master Mix and template DNA.

[0021] The PCR Master Mix comprises: 10 mM ammonium sulfate, 10 mM potassium chloride, 55 mM Tris-HCl at pH 8.3, 2 mM magnesium ions, 0.8 μg / μL BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na₄P₂O₇, 0.2 mM Dntp, and 2 U Taq enzyme.

[0022] The amplification program of the described multiplex amplification kit is as follows: Step 1: Pre-denaturation: 95℃, 1 minute; Thermal cycling: 95℃, 10 seconds, 60℃, 80 seconds, for a total of 30 cycles; Final extension: 60℃, 10 minutes; Incubation: 4℃. The amplification products of this invention need to be subjected to capillary electrophoresis for fragment analysis.

[0023] DNA samples can be derived from one or more of the following: human blood, blood spots, semen, sperm stains, saliva, saliva stains, exfoliated cells, and bone.

[0024] The present invention also provides the application of the above-mentioned multiplex amplification kit in individual identification and paternity testing.

[0025] Beneficial effects

[0026] MiniSTR technology, building upon existing STR analysis techniques, redesigns primers to approximate core repetitive sequences, resulting in smaller PCR amplification product fragments and significantly improving the success rate of typing trace amounts of degraded DNA. By strategically combining highly polymorphic STR loci and designing MiniSTR primers, a multiplex amplification kit targeting autosomal MiniSTRs can be developed, enhancing the individual identification rate of degraded DNA and extremely small samples. This is of significant and practical value in solving the typing challenges of degraded forensic DNA samples and better serving forensic science.

[0027] This invention establishes a fluorescent multiplex amplification kit, which can compensate for the inability to obtain large fragment locus typing in VeriFiler Plus PCR amplification kit and autosomal 30ASTR kit due to sample degradation, thereby increasing the individual identification rate of samples and providing technical support.

[0028] The DNA samples involved in this invention can be derived from human blood, blood spots, semen, sperm spots, saliva, saliva spots, hair, exfoliated cells, bones, etc., and the DNA sources are wide-ranging.

[0029] The beneficial effect of this invention is that during the establishment of a multiplex amplification system, as the number of detected loci increases, the mutual interference between primer pairs also increases. This invention, through optimized design, achieves single-tube amplification of 18 loci, with balanced amplification of loci within each fluorescence channel and between different fluorescence channels, resulting in good genotyping patterns.

[0030] This invention can adapt to the requirement of small sample volume, and can obtain site typing with very small amount of template. The maximum amplification fragment does not exceed 300bp, and the amplification product fragment is relatively small, which is more suitable for amplification detection of trace degraded samples.

[0031] The reagent kit constructed in this invention preferably uses a six-color fluorescent labeling technology, which can be detected on capillary electrophoresis platforms. Attached Figure Description

[0032] Figure 1 This is a diagram showing the amplified locus arrangement of the autosomal MiniSTR kit of this invention.

[0033] Figure 2 The amplification pattern obtained using the amplification system of the present invention.

[0034] Figure 3 , Figure 4 , Figure 5 The amplification pattern obtained using the amplification system of the present invention is shown in the example.

[0035] Figure 6 , Figure 7 , Figure 8 This is a comparison of the amplification patterns of the same sample from the example kit. Detailed Implementation

[0036] To better understand the content of this invention, the following specific embodiment uses genotyping detection of reagent kits in actual samples as a further illustration. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] In this embodiment, the amplification reaction was performed on an ABI Proflex thermal cycler, electrophoresis and detection were performed on an ABI 3500xL genetic analyzer, and data analysis was performed using GeneMapper ID-X software. Other reagents and materials used, such as 36cm POP4, capillary electrophoresis buffer, and Hi-Di, are all conventional materials commonly used by those skilled in the art.

[0038] 1. Sample collection (samples were donated by volunteers)

[0039] 2. DNA extraction

[0040] Genomic DNA was extracted using the Chelex-100 method (refer to Forensic DNA Protocol, Humana Press, 1998). 0.5-5 μL of anticoagulated whole blood or (1-3 mm) * (2-5 mm) blood spots were placed in a 500 μL centrifuge tube. The Chelex solution was vortexed to ensure complete suspension of Chelex. 195 μL of Chelex-100 (5%) solution and 5 μL of proteinase K (20 mg / ml) were added to each tube and vortexed to mix. The mixture was incubated at 56°C for two hours or overnight. After incubation, the tube was shaken for 2 minutes, heated in boiling water for 10 minutes, and then centrifuged at 13000 rpm for 5 minutes. 150 μL of the supernatant was carefully transferred to a new centrifuge tube.

[0041] 3. Reaction system

[0042] After shaking and mixing all reaction reagents (buffer, primer mixture, genomic DNA, etc.), prepare the PCR reaction mixture as follows. The total volume of the amplification system is 10 μL, including 1 μL of primer mixture (10× Primer mix), where the primer concentration is shown in Table 2, 2 μL of reaction buffer (5× Master Mix A), 1 μL of genomic DNA (for template DNA extraction, direct amplification sample, etc.), and 6 μL of ddH2O.

[0043] 4. PCR reaction procedure

[0044] The PCR amplification procedure used in the multiplex amplification kit involved in this invention is shown in Table 6.

[0045] Table 6. Amplification program of the multiplex amplification kit of the present invention.

[0046]

[0047] 5. Capillary electrophoresis detection

[0048] Mix ORG500 internal standard and formamide at a ratio of 2.5:100. Take 12.5 μL of the mixture and add it to a 96-well plate. Then add 1 μL of amplified product sample or allele standard, mix and let stand for a few minutes, denature at 95℃ for 3 minutes, immediately in an ice bath for 3 minutes, centrifuge and place on an ABI 3500xL sequencer for detection.

[0049] 6. Data Analysis

[0050] Import the raw data. On the main page, select "Add sample to project" from the "File" menu, locate the sample file, select the folder, click "add to list," and then click "add." The sample file will then appear in the Project window. Select the analysis parameters. Define the analysis method, panel, and size standard. Browse the raw electrophoresis data of the samples, select the filename of a sample, and select "Raw data" under the "sample" menu. Move the tracking line until the cursor is to the right of the primer peak (before the first orange internal standard peak). Use the value displayed on the X-axis in the lower left corner of the window at this point as the starting point in the analysis method parameters. Click the green "Analyze" button. The "Save Project" dialog box will appear. Name the project and save it. The software will then begin processing the data. After the analysis is complete, the lower left corner will display "Analysis completed." GeneMapper is used. R The ID-X software analyzes the data and generates a graph, such as Figure 3 , Figure 4 , Figure 5 Simultaneously, the autosomal 30A STR kit (Microreader) was used. TM 30AID System) and 36AD STR kit (Microreader) TM The 36A Direct ID System is used for amplification and to generate a spectrum, such as... Figure 6 , Figure 7 , Figure 8 .

[0051] The amplification patterns from the autosomal 30A STR kit and the 36AD STR kit show partial loss of loci or alleles in large fragments, indicating a certain degree of degradation in the sample, making it impossible to obtain genotyping results for all loci. Figure 6 In the D8S1132 variant, the D10S1428 variant was assigned a classification of 24; D10S1435 and D6S477 were not classified. Figure 7 In the 36AD, 12 genotypes were lost at the D10S1435 site. Figure 8The D19S253 locus has a loss of 13 genotypes, the D3S3045 locus has a loss of 13 genotypes, and the D6S477 locus has a loss of 15 genotypes. The kit of this invention precisely fills in the missing loci genotyping. Figure 3 The D8S1132 variant is classified as 20 / 24, the D10S1248 variant as 12 / 15, the D10S1435 variant as 12 and 13, and the D6S477 variant as 11 and 16. This is supplementary. Figure 6 For typing with missing samples, Figure 4 The D10S1435 is classified as 11 / 12, which supplements... Figure 7 Locus typing information of the sample, Figure 5 The D19S253 variant is 11 / 13, the D3S3045 variant is 9 / 13, and the D6S477 variant is 12 / 15, supplementing... Figure 8 The sample lacks a typing.

[0052] The lack of genotyping and loci in the comparison kit reduces the individual identification rate, potentially leading to erroneous judgments and increasing the difficulty of forensic evidence examination. The kit of this invention overcomes the shortcomings of the comparison kit in amplifying degraded samples and missing genotyping sites, ensuring the STR locus typing information of the sample in this case. Furthermore, the amplification time of the kit of this invention is shorter than that of the comparison kit, improving experimental efficiency. This kit also has strong anti-inhibition capabilities, minimizing the impact of inhibitors in difficult samples on amplification.

[0053] This invention established a multiplex amplification system using 17 autosomal STR loci and 1 sex locus. This system can be stably implemented in laboratories equipped with PCR instruments and genetic analysis capabilities, with a detection time of 3-4 hours, saving at least 10-20 minutes compared to control kits. Furthermore, the reagents provided by this invention can be easily manufactured by biotechnology companies and used in biomedical testing institutions, making it suitable for widespread application.

[0054] This invention has been described with reference to specific embodiments thereof. Those skilled in the art will recognize that various modifications or variations can be made to this invention based on the foregoing description, including (but not limited to): changing the fluorophore labeling of different groups, changing the primers for fluorophore labeling (e.g., changing from upstream primers to downstream primers), altering the genomic grouping arrangement according to the allele range of each STR locus, optimizing PCR amplification conditions and primer concentrations according to other PCR reaction systems, and changing the recommended reaction system, etc. It is obvious that the above modifications or variations are possible for those skilled in the art, but these modifications and variations do not depart from the spirit and scope of this invention.

Claims

1. A fluorescent multiplex amplification system for 18 short tandem repeat sequences (MiniSTR), characterized in that, The complex amplification system comprises 17 autosomal STR loci and one gender locus, and contains 18 groups of primer pairs for amplifying the 17 STR loci and the one gender locus, namely CSF1PO, D6S477, D19S253, D8S1132, D5S818, D3S1358, D15S659, D6S1043, D10S1435, Penta E, TPOX, D3S3045, FGA, D7S820, D10S1248, D2S1338, Penta D and the gender locus Amelogenin. The specific amplification primer sequences and concentrations of the 18 loci are as follows: The amplified loci in the complex amplification system are labeled with five colors respectively, and the same fluorescent label is considered as the same group, and the five groups are combined as follows: The first group: CSF1PO, D6S477, D19S253, D8S1132; The second group: Amelogenin, D5S818, D3S1358, D15S659; The third group: D6S1043, D10S1435, Penta E; The fourth group: TPOX, D3S3045, FGA, D7S820; The fifth group: D10S1248, D2S1338, Penta D; The first group is labeled with FAM label, the second group is labeled with HEX label, the third group is labeled with TAMRA label, the fourth group is labeled with ROX label, and the fifth group is PURP.

2. A multiplex amplification kit, characterized by, The complex amplification system of claim 1 is included.

3. The multiplex amplification kit of claim 2, wherein The complex amplification kit further comprises a PCR Master Mix and a DNA template.

4. The multiplex amplification kit of claim 2, wherein The PCR Master Mix comprises 10mM of ammonium sulfate, 10mM of potassium chloride, 55mM of Tris-HCl with pH 8.3, 2mM of magnesium ion, 0.8ug / ul of BSA, 5% of DMSO, 8% of ethylene glycol, 1mM of Na4P2O7 and 0.2mM of Dntp, and 2U of Taq enzyme.

5. The multiplex amplification kit of claim 2, wherein The reaction conditions for amplification of the amplification system are as follows: Step 1: pre-denaturation at 95℃ for 1 minute, Step 2: denaturation at 95℃ for 10 seconds, Step 3: annealing at 60℃ for 80 seconds, a total of 30 cycles; final extension at 60℃ for 10 minutes; Incubation: 4℃.

6. The multiplex amplification kit of claim 2, wherein The DNA sample is derived from one or more of human blood, blood stains, semen, semen stains, saliva, saliva stains, exfoliated cells and bone.

7. The complex amplification kit of any one of claims 2-6 for use in individual identification and paternity identification.

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