Multiplex PCR amplification balancing method suitable for Y-STR site combination and application

Abstract

The application discloses a multiplex PCR amplification balancing method suitable for Y-STR site combination, and belongs to the technical field of forensic genetics and molecular biology. The method is aimed at a multiplex PCR system of 195 non-Y-STR sites, 61 DEG C cooperative annealing temperature is screened out through 58-62 DEG C gradient PCR, two rounds of PCR cycle numbers are optimized as 18 and 16, and the volume ratio of magnetic bead purification is adjusted as 0.75:1 in the first round and 0.65:1 in the second round. Finally, the amplification product is recovered and detected to verify the typing effect. The Y-STR site combination of the application contains 123 autosomes, 70 X-STR sites and two gender sites (Y-indel and Amelogenin), covers long and short fragments, and improves the product abundance imbalance problem under multiplex amplification through multi-parameter cooperative regulation, and is suitable for high-throughput STR typing scenes such as forensic evidence identification and paternity identification.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and forensic genetics, specifically relating to a method for equilibration of multiplex PCR amplification for Y-STR loci without combinations, as well as the application of this method in high-throughput STR typing and related kits. Background Technology

[0002] Short tandem repeat (STR) typing is a core technology for forensic evidence identification and paternity testing. Among these, combinations without Y-STR loci (such as autosomal STR + X-STR) have irreplaceable advantages in mixed sample analysis and female sample identification because they are applicable to samples of all sexes. With the development of high-throughput sequencing technology, multiplex PCR systems with more than 100 loci have become mainstream, but amplification of combinations without Y-STR loci faces significant technical bottlenecks.

[0003] In existing technologies, multiplex PCR reaction conditions are mostly optimized based on systems containing Y-STRs, or only a single parameter (such as annealing temperature or cycle number) is adjusted, without considering the synergistic effects of primer Tm values ​​differences at the 195 sites and amplification kinetics differences between long and short fragments in Y-STR-free systems. Specifically, short fragment STR sites amplify rapidly, easily leading to over-amplification; long fragment STR sites amplify slowly, easily leading to under-amplification, resulting in a significant difference in the abundance of long and short fragment products. Simultaneously, traditional magnetic bead purification parameters are not optimized for fragment distribution in Y-STR-free systems, easily causing loss of short fragment products or insufficient purification of long fragment products, further exacerbating amplification imbalance.

[0004] This amplification imbalance leads to uneven signal distribution in sequencing maps, affecting genotyping interpretation. Although existing technologies have proposed balancing strategies for multiplex PCR, such as primer concentration adjustment and annealing temperature optimization, none have achieved coordinated control of the three parameters of "temperature-cycle number-purification" for the specific system with "195 Y-STR-free sites," thus failing to fundamentally alleviate the mismatch in amplification abundance between long and short fragments. Therefore, there is an urgent need to develop a multi-parameter coordinated amplification process adapted to this site system. Summary of the Invention

[0005] (a) Purpose of the invention This invention addresses the technical problem of uneven amplification abundance of long and short STR fragments in existing multiplex PCR systems without Y-STR sites by providing a method for synergistic regulation of amplification balance using three parameters: temperature, cycle number, and purification. It also provides the application and kit for this method. By simultaneously regulating these three sets of process parameters, the difference in product abundance between different STR fragments within the system can be reduced.

[0006] (II) Technical Solution To achieve the above objectives, the present invention adopts the following technical solution: A method for equilibration of multiplex PCR amplification adapted to combinations without Y-STR sites includes the following steps: S1: Gradient PCR annealing temperature screening: For multiplex PCR systems containing multiple non-Y-STR sites, gradient PCR experiments were performed with an annealing temperature gradient of 55~65℃. The co-annealing temperature for the absence of Y-STR sites was screened to be 60~62℃. S2: Optimization of the number of PCR cycles in two rounds: Using the synergistic annealing temperature, the number of PCR cycles in the first round is set to 15-20, and the number of PCR cycles in the second round is set to 14-18; S3: Optimization of magnetic bead purification parameters: The two rounds of PCR amplification products were purified by magnetic beads. The volume ratio of magnetic beads to PCR products in the first round was 0.7~0.8:1, and the volume ratio of magnetic beads to PCR products in the second round was 0.6~0.7:1. S4: Amplification product recovery and detection: Collect the purified amplification products and perform product detection using capillary electrophoresis or high-throughput sequencing.

[0007] Preferably, the non-Y-STR site combination is a combination of autosomal STR sites and X-STR sites, covering short fragments (≤200bp) and long fragments (≥300bp) sites, with a fragment length span of ≥150bp. The number of non-Y-STR sites is 160~200, of which 100~125 are autosomal STR sites and 60~80 are X-STR sites.

[0008] Preferably, the reaction system for the gradient PCR experiment comprises: 1×PCR buffer, 1.5~2.5 mM Mg²⁺, 150~250 μM dNTPs, 0.1~0.3 μM primers at each site, 0.8~1.2 U high-fidelity DNA polymerase, 1~10 ng template DNA, and water added to a final volume of 20~30 μL.

[0009] Preferably, the thermal cycling program for the gradient PCR experiment and two rounds of PCR amplification is as follows: initial denaturation at 94~96℃ for 3~5 min; denaturation at 94~96℃ for 25~35 s, annealing at 60~62℃ for 40~50 s, extension at 70~74℃ for 25~35 s, followed by amplification according to the optimized cycle number; final extension at 70~74℃ for 6~8 min; and incubation at 2~6℃.

[0010] Preferably, the operating conditions for the magnetic bead purification are as follows: incubation at room temperature for 8-12 min, magnetic rack adsorption for 4-6 min, washing twice with 65-75% ethanol, magnetic rack adsorption for 2-4 min after each wash, drying at room temperature for 2-6 min, and elution of the amplification product with TE buffer.

[0011] Preferably, the pH value of the TE buffer is 7.8~8.2. During elution, 12~18 μL of TE buffer is added to the dried magnetic beads, incubated at room temperature for 4~6 min, and then the supernatant is collected after magnetic adsorption for 4~6 min to obtain the purified amplification product.

[0012] Preferably, the high-fidelity DNA polymerase is a DNA polymerase with 3'→5' exonuclease activity, and the temperature fluctuation during amplification is controlled within the range of ±0.3~±0.7℃.

[0013] One method is applied to high-throughput STR typing without Y-STR locus combinations, with applications in forensic evidence identification, paternity testing, and human individual identification.

[0014] A multiplex PCR amplification kit without Y-STR site combinations comprises the optimized reaction conditions specification of the method described in claim 1, PCR reaction reagents, and magnetic bead purification reagents; the optimized reaction conditions specify a co-annealing temperature of 60-62°C, two PCR cycles of 15-20 and 14-18, respectively, and a magnetic bead to PCR product volume ratio of 0.7-0.8:1 and 0.6-0.7:1, respectively; the PCR reaction reagents comprise PCR buffer, Mg²⁺, dNTPs, a mixture of 160-200 sets of non-Y-STR site primers, and high-fidelity DNA polymerase; the magnetic bead purification reagents comprise magnetic bead suspension, 65-75% ethanol washing buffer, and TE buffer.

[0015] Preferably, the kit also includes individually packaged positive and negative controls. The positive control is a human genomic DNA containing standard Y chromosome-free DNA at a concentration of 1-10 ng / μL. The negative control is a sterile, enzyme-free TE buffer. All reagent components are individually packaged in single tubes and stored at low temperature in the dark.

[0016] (III) Beneficial Effects Compared with the prior art, the present invention has the following significant advantages: 1. This invention simultaneously limits three sets of linked process parameters: annealing temperature, amplification cycle, and magnetic bead purification. Unlike existing operation schemes that only adjust a single parameter, this invention can reduce the difference in product abundance between long and short STR fragments within the same multi-stage system.

[0017] 2. Differentiated magnetic bead purification adapts to the fragment distribution characteristics of the two rounds of amplification products, reducing the loss of long-fragment nucleic acid products and resulting in a more uniform distribution of amplification products within the system.

[0018] 3. The entire process relies on conventional PCR and magnetic bead purification equipment, without the need for specially customized instruments; the matching reagent kit can be standardized and packaged, suitable for batch forensic sample testing, and has industrial application feasibility. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Screening and Determination of Synergistic Annealing Temperature Range 1.1 Experimental Materials and Systems A primer mixture of 195 non-Y-STR loci (containing 123 autosomal loci, 70 X-STR loci, and 2 sex loci, covering short fragments ≤200bp, long fragments ≥300bp, and a length span ≥150bp) was selected; standard human genomic DNA (9947A, concentration 5ng / μL); 1×PCR buffer (containing 1.5~2.5mM Mg²⁺ adjustable), 200μM dNTPs, 0.2μM primers for each locus, and 1.0U high-fidelity DNA polymerase (with 3'→5' exonuclease activity) was added to a final volume of 25μL to construct a gradient PCR reaction system.

[0021] 1.2 Experimental Methods Annealing temperature gradients were set at 55℃, 58℃, 60℃, 61℃, 62℃, and 65℃. The thermal cycling program was uniform: initial denaturation at 95℃ for 4 min; denaturation at 95℃ for 30 s, gradient temperature annealing for 45 s, extension at 72℃ for 30 s, for a total of 18 cycles; final extension at 72℃ for 7 min; and holding at 4℃. After amplification, the results were analyzed using an ABI 3730xl capillary electrophoresis system. The amplification uniformity score, short fragment over-amplification rate, long fragment under-amplification rate, and primer dimerization rate were statistically analyzed for 120 loci at each temperature. Each experiment was repeated three times, and the average value was taken.

[0022] 1.3 Experimental Data and Analysis Data Analysis: At 55~58℃, the amplification uniformity score was below 79, with obvious over-amplification of short fragments and under-amplification of long fragments. Within the 60~62℃ range, the amplification uniformity score was ≥90, and the rates of short fragment over-amplification, long fragment under-amplification, and primer dimer occurrence were all at low levels. At 65℃, the amplification uniformity score decreased, and the binding efficiency of long-fragment primers decreased. Conclusion: 60~62℃ is the optimal annealing range for this system, and 61℃ is the optimal temperature for a single group.

[0023] II. Example 2: Co-optimization of the number of PCR cycles in two rounds 2.1 Experimental Methods Based on the synergistic annealing temperature of 61℃ determined in Example 1, cross-combination experiments were set up with first-round cycle numbers (15, 16, 18, 19, 20) and second-round cycle numbers (14, 15, 16, 17, 18), for a total of 15 groups. The remaining reaction system and thermal cycling program parameters were consistent with those in Example 1. After amplification, purification was performed using 0.75:1 (first round) / 0.65:1 (second round) magnetic beads. The difference in amplification efficiency between long and short fragments was detected by capillary electrophoresis (measured by the standard deviation of the peak area ratio of short fragments to long fragments). Each group was repeated 3 times.

[0024] 2.2 Experimental Data and Analysis Data Analysis: Groups 15 / 14 and 20 / 18: The difference in amplification efficiency between long and short fragments was >14%, and the peak regularity score was <8.6. This was attributed to insufficient amplification of long fragments due to too few cycles, or excessive enrichment of short fragments due to too many cycles. Group 18 / 16: The difference in amplification efficiency between long and short fragments decreased to 10.5±0.6%, the peak regularity score reached 9.3, and the allele detection rate was 99.6%, with all indicators being the best among all groups; Group 19 / 17: The amplification efficiency difference was slightly higher than that of Group 18 / 16, attributed to the increased number of cycles in the second round leading to redundancy in short fragment amplification and slightly poorer balance. Conclusion: The optimal number of cycles in the first round (15-20 cycles) and the second round (14-18 cycles) of this invention is within the optimal range, with 18 / 16 cycles being the optimal parameter combination, which can achieve a precise balance between long and short fragment amplification.

[0025] III. Example 3: Collaborative Optimization of Magnetic Bead Purification Parameters 3.1 Experimental Methods Based on the optimal temperature (61℃) and cycle number (18 / 16), a gradient of magnetic bead to PCR product volume ratios was established: first round (0.7:1, 0.73:1, 0.75:1, 0.78:1, 0.8:1), second round (0.6:1, 0.63:1, 0.65:1, 0.68:1, 0.7:1), for a total of 25 groups. The purification procedure was standardized as follows: incubation at room temperature for 8–12 min, magnetic adsorption for 4–6 min, washing twice with 70% ethanol (adsorption for 2–4 min after each wash), drying at room temperature for 2–6 min, and elution with 15 μL of TE buffer (pH 7.8–8.2). Product recovery rate, short fragment retention rate, long fragment purification efficiency, and impurity removal rate were statistically analyzed, with each group repeated three times.

[0026] 3.2 Experimental Data and Analysis Data Analysis: 0.7:1 / 0.6:1 group: product recovery rate <89%, short fragment retention rate 92.5%, long fragment purification efficiency <86%, which is attributed to insufficient adsorption of short fragments and residual purification of long fragments due to insufficient magnetic bead dosage; The 0.75:1 / 0.65:1 group showed the following optimal results: product recovery rate of 92.8±1.0%, short fragment retention rate of 96.1%, long fragment purification efficiency of 91.2%, and impurity removal rate of 85.6%. The 0.8:1 / 0.7:1 group showed an impurity removal rate of only 78.5%, attributed to excessive magnetic bead usage leading to increased adsorption of non-specific impurities and a slight decrease in long fragment purification efficiency. Conclusion: The optimal purification range for this invention is a magnetic bead volume ratio of 0.7~0.8:1 in the first round and 0.6~0.7:1 in the second round, with 0.75:1 / 0.65:1 being the optimal parameter combination, balancing product recovery, fragment retention, and impurity removal.

[0027] IV. Example 4: Practical Application Validation and Performance Summary of the Reagent Kit 4.1 Experimental Materials The kit of this invention comprises: ① an optimized reaction condition instruction manual (indicating annealing temperature of 60-62℃, cycle number of 15-20 / 14-18, and purification ratio of 0.7-0.8 / 0.6-0.7); ② PCR reaction reagents (1×PCR buffer, 2.0mM Mg²⁺, 200μM dNTPs, a mixture of 120 Y-STR site-free primers, and 1.0U high-fidelity DNA polymerase); ③ magnetic bead purification reagents (magnetic bead suspension, 70% ethanol washing buffer, and TE buffer pH 8.0); ④ positive control (5ng / μL standard 9947A DNA) and negative control (sterile enzyme-free TE buffer); all reagents are individually aliquoted and stored at -20℃ in the dark.

[0028] 4.2 Experimental Methods Twenty actual forensic samples were selected, covering five special scenarios: ① trace samples (bloodstains <10μL, saliva stains <5μL, n=6); ② degraded samples (old bones, hair, n=5); ③ mixed female samples (female bloodstains + male semen, n=4); ④ routine bloodstain samples (n=3); ⑤ blank control (sterile water, n=2). The procedure was performed according to the kit instructions and the method described in claim 1. Amplification temperature fluctuations were controlled within ±0.5℃. After amplification, capillary electrophoresis was performed to detect allele detection rate, locus amplification uniformity, genotyping success rate, and negative control amplification results.

[0029] 4.3 Experimental Data and Analysis Comprehensive data analysis: All 20 test samples yielded interpretable capillary electrophoresis patterns, while the two blank controls showed no specific amplification products.

[0030] 4.4 Overall Summary This invention utilizes three sets of matching process parameters—a synergistic annealing temperature of 60–62°C, two cycles of 15–20 / 14–18, and purification with magnetic beads of 0.7–0.8 / 0.6–0.7—to reduce the difference in amplification product abundance between long and short STR fragments in a multiplex PCR system lacking ultra-large Y-STR loci. Gradient experimental data show that annealing at 61°C, 18 cycles in the first round and 16 cycles in the second, and a magnetic bead ratio of 0.75:1 / 0.65:1 are the optimal matching parameters for this system. Actual forensic sample testing yielded effective genotyping profiles, and the blank control showed no amplification contamination. The entire process employs industry-standard molecular experimental equipment, and the accompanying reagent kits can be standardized and packaged for production.

[0031] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications and substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

[0032] Appendix 1. Primer Design Instructions for the Reagent Kit

Claims

1. A method for equilibration of multiplex PCR amplification adapted to combinations without Y-STR sites, characterized in that, Includes the following steps: S1: Gradient PCR annealing temperature screening: For multiplex PCR systems containing multiple non-Y-STR sites, a gradient annealing temperature of 55~65℃ was set for gradient PCR experiments, and the co-annealing temperature of the non-Y-STR sites was screened to be 60~62℃. S2: Optimization of the number of PCR cycles in two rounds: Using the synergistic annealing temperature, the number of PCR cycles in the first round is set to 15-20, and the number of PCR cycles in the second round is set to 14-18; S3: Optimization of magnetic bead purification parameters: The two rounds of PCR amplification products were purified by magnetic beads. The volume ratio of magnetic beads to PCR products in the first round was 0.7~0.8:1, and the volume ratio of magnetic beads to PCR products in the second round was 0.6~0.7:

1. S4: Amplification product recovery and detection: Collect the purified amplification products and perform product detection using capillary electrophoresis or high-throughput sequencing.

2. The method according to claim 1, characterized in that, The non-Y-STR site combinations are mainly combinations of autosomal STR sites and X-STR sites, covering short fragments (≤200bp) and long fragments (≥300bp) sites, with a fragment length span of ≥150bp. The number of non-Y-STR sites is 160~200, of which 100~125 are autosomal STR sites and 60~80 are X-STR sites. The amplification primer sequences for each site are shown in Appendix 1.

3. The method according to claim 1, characterized in that, The reaction system for the gradient PCR experiment includes: 1×PCR buffer, 1.5~2.5 mg²⁺, 150~250 μM dNTPs, 0.1~0.3 μM primers at each site, 0.8~1.2 U high-fidelity DNA polymerase, 1~10 ng template DNA, and water added to a final volume of 20~30 μL.

4. The method according to claim 1, characterized in that, The thermal cycling program for the gradient PCR experiment and two rounds of PCR amplification is as follows: initial denaturation at 94~96℃ for 3~5 min; denaturation at 94~96℃ for 25~35 s, annealing at 60~62℃ for 40~50 s, extension at 70~74℃ for 25~35 s, followed by amplification according to the optimized cycle number; final extension at 70~74℃ for 6~8 min; and incubation at 2~6℃.

5. The method according to claim 1, characterized in that, The purification conditions for the magnetic beads are as follows: incubation at room temperature for 8-12 min, magnetic rack adsorption for 4-6 min, washing twice with 65-75% ethanol, magnetic rack adsorption for 2-4 min after each wash, drying at room temperature for 2-6 min, and elution of the amplification product with TE buffer.

6. The method according to claim 5, characterized in that, The pH value of the TE buffer is 7.8~8.

2. During elution, 12~18μL of TE buffer is added to the dried magnetic beads, incubated at room temperature for 4~6 min, and then the supernatant is collected after magnetic adsorption for 4~6 min, which is the purified amplification product.

7. The method according to claim 1, characterized in that, The high-fidelity DNA polymerase is a DNA polymerase with 3'→5' exonuclease activity, and the temperature fluctuation during amplification is controlled within the range of ±0.3~±0.7℃.

8. The application of the method according to any one of claims 1 to 7 in high-throughput STR genotyping without Y-STR locus combinations, characterized in that, Application scenarios include forensic evidence identification, paternity testing, and human individual identification.

9. A multiplex PCR amplification kit without Y-STR site combinations, characterized in that, The method comprises an optimized reaction condition specification, PCR reaction reagents, and magnetic bead purification reagents according to claim 1. The optimized reaction conditions include a synergistic annealing temperature of 60-62°C, two PCR cycles of 15-20 and 14-18, and a magnetic bead to PCR product volume ratio of 0.7-0.8:1 and 0.6-0.7:1, respectively. The PCR reaction reagents include PCR buffer, Mg²⁺, dNTPs, a mixture of 160-200 sets of primers for non-Y-STR sites, and high-fidelity DNA polymerase. The magnetic bead purification reagents include magnetic bead suspension, 65-75% ethanol washing buffer, and TE buffer. Primer sequences are provided in Appendix 1.

10. The reagent kit according to claim 9, characterized in that, The kit also includes individually packaged positive and negative controls. The positive control contains standard Y chromosome-free human genomic DNA at a concentration of 1-10 ng / μL. The negative control is a sterile, enzyme-free TE buffer. All reagent components are individually packaged in single tubes and stored at low temperature in the dark.

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