CN111718984A - Method for STR typing of forensic mixed DNA sample - Google Patents
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Abstract
The invention discloses a method for STR typing of a forensic mixed DNA sample. The invention provides a method for STR typing of a biological sample, which comprises the following steps: extracting genome DNA of a biological sample, carrying out micro-droplet digital PCR amplification by using the genome DNA as a template, sequencing an amplification product, and carrying out STR typing according to a sequencing result. The biological sample is a mixed biological sample. The invention also provides a method for performing STR typing on the DNA sample, which comprises the following steps: and (3) carrying out micro-droplet digital PCR amplification by taking the DNA sample as a template, sequencing the amplified product, and carrying out STR typing according to a sequencing result. The DNA sample is a mixed DNA sample. The invention has great application prospect in forensic DNA inspection.
Description
Technical Field
The invention relates to a method for STR typing of a forensic mixed DNA sample.
Background
With the increase of DNA test level, micro-contact type materials become the main materials for DNA laboratories, and most of the materials obtained from such tests are mixed DNA typing (such as mixing of DNA of a victim and a suspect, mixing of 2 or more samples of the suspect, or mixing of the suspect and another unrelated individual, etc.). It is precisely these mixed samples that are of great practical significance in providing case clues, establishing the scope of investigation and providing forensic evidence. However, the mixed sample DNA atlas has many STR peak spectra, complex banding patterns and unstable peak patterns due to different mixing numbers and mixing ratios, which results in difficult analysis and high qualitative risk of the mixed sample, so that the mixed sample is easily discarded or ignored, and important case information is omitted or the DNA evidence efficacy is greatly reduced.
At present, the inspection and analysis method for the mixed DNA sample by the national and international legal medical community mainly comprises the steps of splitting the mixed DNA atlas based on computer software, or manually splitting by experts, or separating single cells of the mixed DNA sample so as to obtain single DNA typing, thereby separating valuable DNA information from the mixed sample. The single cell separation technique relies on specialized micromanipulation equipment and trained operators, and conditions under which single cell separation can be performed are limited. However, manual separation based on computer technology or experienced experts relies on obtaining a high-quality mixed map, and the initial state of DNA mixing in a test material cannot be changed. However, in the actual case, the material itself may be mixed with the background of the DNA of the victim, the DNA content of the suspect is relatively low, and the low-content DNA template is submerged in the sea with the high-content DNA template.
The digital PCR technology is that the sample is micro-drizzled by a micro-drop generator and is 'split' into 2 × 10 by water-in-oil technology4The PCR reaction was carried out after 1nL volume of each microdropletAn independent PCR reaction system, the method has high flux, simple operation and low cost.
Disclosure of Invention
The invention aims to provide a method for STR typing of a forensic mixed DNA sample.
The invention provides a method for STR typing of a biological sample, which comprises the following steps: extracting genome DNA of a biological sample, carrying out micro-droplet digital PCR amplification by using the genome DNA as a template, sequencing an amplification product, and carrying out STR typing according to a sequencing result. The biological sample is a mixed biological sample, for example, a mixed blood sample derived from 2 or more individuals, a mixed cell sample derived from 2 or more individuals, or the like. The method is used in the field of forensic identification. The reaction procedure of the micro-drop digital PCR amplification is as follows: 1min at 95 ℃; 29 cycles of 94 ℃ for 30s and 59 ℃ for 1.5 min; 10min at 60 ℃; 10min at 98 ℃. The temperature rising and falling speed of the micro-drop type digital PCR amplification is set to be 2 ℃/s. The reaction system of the micro-drop digital PCR amplification (20 μ L): 2 μ L template, 2 μ L
Primer Set、6μL
Master Mix and 10. mu.L ddPCR Super Mix for probes (no dUTP). The sequencing employs a genetic analyzer. Is concretely made by
And performing STR typing by using ID-X analysis software.
The invention also provides a method for performing STR typing on the DNA sample, which comprises the following steps: and (3) carrying out micro-droplet digital PCR amplification by taking the DNA sample as a template, sequencing the amplified product, and carrying out STR typing according to a sequencing result. The DNA sample is a mixed DNA sample, that is, a DNA sample derived from 2 or more individuals, for example, a DNA sample derived from a mixed blood sample of 2 or more individuals, a DNA sample derived from a mixed cell sample of 2 or more individuals, or the like. The method is used in the field of forensic identification. Said droplet digital PCR amplificationThe reaction procedure is as follows: 1min at 95 ℃; 29 cycles of 94 ℃ for 30s and 59 ℃ for 1.5 min; 10min at 60 ℃; 10min at 98 ℃. The temperature rising and falling speed of the micro-drop type digital PCR amplification is set to be 2 ℃/s. The reaction system of the micro-drop digital PCR amplification (20 μ L): 2 μ L template, 2 μ L
Primer Set、6μL
Master Mix and 10. mu.L ddPCR Super Mix for probes (no dUTP). The sequencing employs a genetic analyzer. Is concretely made by
And performing STR typing by using ID-X analysis software.
The invention also provides a system for performing STR typing on a biological sample, which is characterized by comprising the following steps: comprises a component A for extracting DNA, a component B for carrying out micro-drop digital PCR amplification and a component C for sequencing. The component B is a microdroplet generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The component C is a genetic analyzer. The biological sample is a mixed biological sample, for example, a mixed blood sample derived from 2 or more individuals, a mixed cell sample derived from 2 or more individuals, or the like. The STR typing is STR typing in the field of forensic identification.
The invention also protects a system for STR typing of DNA samples, which is characterized in that: comprises a component B for carrying out the micro-drop digital PCR amplification and a component C for carrying out the sequencing. The component B is a microdroplet generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The component C is a genetic analyzer. The DNA sample is a mixed DNA sample, that is, a DNA sample derived from 2 or more individuals, for example, a DNA sample derived from a mixed blood sample of 2 or more individuals, a DNA sample derived from a mixed cell sample of 2 or more individuals, or the like. The STR typing is STR typing in the field of forensic identification.
The invention also protects the application of the component A for DNA extraction, the component B for micro-drop digital PCR amplification and the component C for sequencing in the preparation of a system for STR typing of biological samples. The component B is a microdroplet generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The component C is a genetic analyzer. The biological sample is a mixed biological sample, for example, a mixed blood sample derived from 2 or more individuals, a mixed cell sample derived from 2 or more individuals, or the like. The STR typing is STR typing in the field of forensic identification.
The invention also protects the application of the component B for carrying out the micro-drop digital PCR amplification and the component C for carrying out the sequencing in the preparation of a system for carrying out STR typing on DNA samples. The component B is a microdroplet generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The component C is a genetic analyzer. The DNA sample is a mixed DNA sample, i.e., a DNA sample derived from more than 2 individuals, such as mixed blood derived from more than 2 individualsA sample DNA, a DNA sample derived from a mixed cell sample of 2 or more individuals, and the like. The STR typing is STR typing in the field of forensic identification.
The invention also protects the use of the assembly for performing microdroplet digital PCR amplification in STR typing of biological samples. The STR typing is STR typing in the field of forensic identification. The components for carrying out the micro-drop type digital PCR amplification are a micro-drop generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The biological sample is a mixed biological sample, for example, a mixed blood sample derived from 2 or more individuals, a mixed cell sample derived from 2 or more individuals, or the like.
The invention also protects the application of the assembly for carrying out the micro-drop digital PCR amplification in the STR typing of the DNA sample. The STR typing is STR typing in the field of forensic identification. The components for carrying out the micro-drop type digital PCR amplification are a micro-drop generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
PrimerSet and
master Mix. The DNA sample is a mixed DNA sample, that is, a DNA sample derived from 2 or more individuals, for example, a DNA sample derived from a mixed blood sample of 2 or more individuals, a DNA sample derived from a mixed cell sample of 2 or more individuals, or the like.
The invention also protects the application of the assembly for carrying out the micro-drop digital PCR amplification in the preparation of a system for STR typing of biological samples. The STR typing is STR typing in the field of forensic identification. The components for carrying out the micro-drop type digital PCR amplification are a micro-drop generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The biological sample is a mixed biological sample, for example, a mixed blood sample derived from 2 or more individuals, a mixed cell sample derived from 2 or more individuals, or the like.
The invention also protects the application of the assembly for carrying out the micro-droplet digital PCR amplification in the preparation of a system for STR typing of DNA samples. The STR typing is STR typing in the field of forensic identification. The components for carrying out the micro-drop type digital PCR amplification are a micro-drop generator, a gradient PCR amplification instrument, a ddPCR Super Mix for probes (no dUTP),
Primer Set and
master Mix. The DNA sample is a mixed DNA sample, that is, a DNA sample derived from 2 or more individuals, for example, a DNA sample derived from a mixed blood sample of 2 or more individuals, a DNA sample derived from a mixed cell sample of 2 or more individuals, or the like.
Any of the above genetic analyzers is 3500XL genetic analyzer manufactured by Applied Biosystems.
Any one of the above gradient PCR amplification apparatuses is a T100 type gradient PCR apparatus manufactured by Bio-Rad of America.
Preparing DNA standard substances such as 9947A, 007 and the like into DNA mixed samples with different mixing ratios, collecting mixed DNA detection materials in an actual case, respectively using traditional PCR amplification and micro-droplet digital PCR amplification, and detecting amplification products by a genetic analyzer and analyzing STR (short tandem repeat) patterns. By comparing the amplification effects of the two methods, the amplification result of the droplet digital PCR on the mixed DNA sample is better. The STR peak spectrum of 007 can be better detected when the mixing ratio of 9947A to 007 is as low as 20:1, and a DNA mixed spectrum with better quality can be obtained in a mixed DNA sample of an actual case. The micro-drop digital PCR technology can improve the amplification effect of low-concentration components in mixed DNA, weaken the amplification preference effect, facilitate the interpretation of DNA mixed maps and have good application potential in forensic mixed DNA inspection.
To date, ddPCR technology has not been applied to mixed DNA amplification and analysis in the forensic field, and is a technical gap. The invention utilizes the characteristic that the micro-drop type digital PCR water-in-oil can generate 2 ten thousand micro-drops, changes a reaction system, adjusts reaction parameters, weakens the amplification preference of DNA, can improve the amplification probability of low-component DNA in a mixed sample, adjusts the mixing proportion of DNA products, is more beneficial to DNA atlas analysis, deeply excavates the information of the mixed DNA sample, and provides a new idea and a new method for mixed DNA inspection. The method provided by the invention can ensure that the low-content DNA template can obtain equal amplification opportunities, has higher amplification sensitivity and precision, and can detect the low-component mixture with the mixing ratio of more than 5% (20: 1). The invention has great application prospect in forensic DNA inspection.
Drawings
FIG. 1 shows the results of conventional PCR amplification at 9947: 007: 1.
FIG. 2 shows the results of conventional PCR amplification at 9947: 007: 2: 1.
FIG. 3 shows the results of conventional PCR amplification at 9947: 007: 5: 1.
FIG. 4 shows the results of conventional PCR amplification at 9947: 007: 10: 1.
FIG. 5 shows the results of conventional PCR amplification at 9947: 007: 20: 1.
FIG. 6 shows the results of conventional PCR amplification at 9947: 007: 50: 1.
FIG. 7 shows the result of ddPCR amplification when 9947: 007: 1.
FIG. 8 shows the result of ddPCR amplification at 9947: 007: 2: 1.
FIG. 9 shows the result of ddPCR amplification when 9947: 007: 5: 1.
FIG. 10 shows the result of ddPCR amplification when 9947: 007: 10: 1.
FIG. 11 shows the result of ddPCR amplification at 9947: 007: 20: 1.
FIG. 12 shows the result of ddPCR amplification at 9947: 007: 50: 1.
FIG. 13 shows the results of conventional amplification of sample No. 9.
FIG. 14 shows the result of ddPCR amplification of sample No. 9.
FIG. 15 shows the results of conventional amplification of sample No. 10.
FIG. 16 shows the result of ddPCR amplification of sample No. 10.
FIG. 17 shows the results of conventional amplification of sample No. 12.
FIG. 18 shows the result of ddPCR amplification of sample No. 12.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
STRs (short distance repeats) are widely present in the genomes of humans and mammals and are highly polymorphic, generally consisting of a core sequence of 2-6 nucleotides, which is arranged in tandem repeats and whose length polymorphism results from a change in the number of repeats of the core sequence. The number of repeats of a repeat sequence at a particular position on a chromosome is fixed for a particular individual and may vary from individual to individual at the same position, constituting polymorphisms of these repeat sequences in the population.
9947A human genome DNA standard (9947A standard for short), female, 1 ng/ul, is a product of Shanghai Boliter Biotechnology Co., Ltd, the catalog number of the product is AM100035, and the product link is http:// www.biosun.cn/news/7718. htm.
007 human genomic DNA Standard (007 Standard for short), Male, 0.1 ng/. mu.l, from Thermo
The Kit comprises components in the PCR Amplification Kit, wherein the catalog number of the Kit is 4476135, and the product link of the Kit is https:// assets.qa4477596.pdf。
The PCR Amplification Kit also has components
Primer Set and Module
Master Mix。
AmpFLSTRTMIdentifilerTMPCR Amplification Kit, product of Thermo corporation, catalog number 4322288, product Link
https://www.thermofisher.com/order/catalog/product/4322288？SID＝srch-srp-4322288。
The Bio-Rad QX200 droplet digital PCR system, manufactured by Bio-Rad corporation, USA, comprises a QX200 droplet generator and a QX200 droplet analyzer, and only the droplet generator is used in the examples. The gradient PCR amplification instrument is a T100 type gradient PCR instrument manufactured by the company of Bio-Rad in the United states. The genetic analyzer is 3500XL type genetic analyzer manufactured by Applied Biosystems,
the ID-X analysis software is matched with the ID-X analysis software.
ddPCR Super Mix for probes (no dUTP), BIO-RAD, catalog number 1863023, product Link
http://www.bio-rad.com/en-us/sku/1863023-ddpcr-supermix-for-probes-no-dutp？ID＝1863023。
By using
The allele genotypes of the respective loci of the 9947A and 007 standards were detected by PCR Amplification Kit and the procedures described in the specification, respectively, and the results are shown in Table 1. Information on the individual alleles of individual STR loci is found in the STRbase database (website https:// base. nist. gov/index. htm). Amelogenin gene, i.e., Amelogenin gene, having one in human X chromosomeThe artificial tooth enamel gene (AMG) is a tooth enamel gene (AMGL) near the centromere of the human Y chromosome, the AMG has 90% homology with the AMGL sequence, X represents a characteristic peak of the AMG, and Y represents a characteristic peak of Y. In table 1, filled in each cell are the genotypes of the two chromosomes, separated by a comma. In Table 1, -represents no amplification product of the corresponding locus.
TABLE 1
Example 1 detection of Standard Mixed sample
First, preparation of mixed sample
The proportions of the 6 mixed samples are shown in Table 2.
TABLE 2
Second, conventional PCR amplification
By using
PCR Amplification Kit was performed as described.
The reaction system is 20 μ L, and consists of 2 μ L template and 2 μ L
Primer Set、6μL
Master Mix and 10. mu.L ddH2And (C) O.
The templates were 6 mixed samples prepared in step one.
The reaction procedure is described in the kit instructions, 29 cycles.
Three, micro-drop digital PCR amplification
The reaction system is 20 μ L, and consists of 2 μ L template and 2 μ L
Primer Set、6μL
Master Mix and 10. mu.L ddPCR Super Mix for probes (no dUTP).
The templates were 6 mixed samples prepared in step one.
1. mu.L of the reaction system and 70. mu.L of the microdroplet-forming oil were applied to the microdroplet-generating card, covered with a rubber pad and placed in the microdroplet-generating apparatus to form microdroplets.
2. Transferring the microdroplets of about 40 mu L obtained in the step 1 into a 96-well plate, sealing the microdroplets by using a heat sealing membrane instrument, and then placing the microdroplets in a T100 type gradient PCR amplification instrument for reaction. Reaction procedure: 1min at 95 ℃; 29 cycles of 94 ℃ for 30s and 59 ℃ for 1.5 min; 10min at 60 ℃; 10min at 98 ℃; infinity at 15 ℃; the temperature rise and fall speed is set to be 2 ℃/s. The product was stored at 4 ℃.
3. And (3) centrifuging the product obtained in the step (2), primarily removing the oil phase at the bottom of the tube, adding 10 mu L of TE solution, adding 40 mu L of chloroform into a fume hood, mixing the solution up and down, transferring the mixture into a 1.5ml centrifuge tube with a cover, carrying out vortex oscillation at the highest speed for 1 minute, centrifuging the mixture for 10 minutes at 15000g, removing the upper chloroform phase, and transferring the upper chloroform phase into a new 1.5ml centrifuge tube.
IV, electrophoretic analysis
The product of step two or the product of step three was loaded on a genetic analyzer for detection (loading amount 1. mu.L). By using
STR typing was carried out by ID-X analysis software (V3.2) with the RFU value of the fluorescence peak set at 50 or more.
The results of the conventional post-PCR electrophoretic analysis of 6 pooled samples are shown in FIGS. 1-6. When the ratio of female to male components in the mixed sample is 5:1, pooled STR peak profiles can also be barely detected and manual resolution of pooled samples is basically possible (fig. 3), but when the female to male component ratio in the pooled sample becomes 10:1, extremely low Y peak can be detected from sex locus Amel, but it is difficult to split 007 allele at other loci (FIG. 4), and when the mixing ratio is increased to 20:1 or even 50:1, only 9947 single type is left from STR peak spectrum judgment, and mixed components are difficult to find (FIG. 5, FIG. 6).
The results of electrophoretic analysis after the 6 mixed sample droplet digital PCR amplification are shown in fig. 7 to 12. When the mixing ratio of male and female is 1:1 and 1:2 (figure 7, figure 8), the effect difference from the conventional PCR is not obvious, but when the mixing ratio is increased to 5:1, the result of the micro-drop digital PCR amplification is obviously better than that of the conventional PCR, the mixing of male components can be clearly seen, the STR locus of a male can be clearly separated to obtain a single typing (figure 9), and when the mixing ratio is 10:1, the male components can still be confirmed and separated by the mixed map (figure 10). Even when the mixing ratio is increased to 20:1 and 50:1, male components can still be detected at the Amel locus, which indicates that the sample is a mixed sample, Y-STR typing can be continuously carried out without missing or misjudging the information of the mixed pattern, and STR typing of the male components can still be obtained on partial loci (FIG. 11, FIG. 12). Therefore, the quality of the mixed spectrum is obviously improved by using the micro-drop digital PCR amplification instead.
Example 2 detection of actual samples
Supplying a sample book: blood sample mixture (sample No. 12), exfoliated cell mixture (sample No. 9, sample No. 10) from the central routine.
Firstly, extracting the genome DNA of each sample to be tested respectively.
Second, conventional PCR amplification
Using AmpFLSTRTMIdentifilerTMPCR Amplification Kit was performed as described.
The amount of genomic DNA contained in each reaction system was 0.5 ng.
The templates are the genomic DNA of each test sample prepared in the first step.
Three, micro-drop digital PCR amplification
The reaction system was 20. mu.L consisting of 2. mu.L of template (DNA content in 2. mu.L of template: 0.5ng)、2μL
Primer Set、6μL
Master Mix and 10. mu.L ddPCR Super Mix probes (no dUTP).
The templates are the genomic DNA of each test sample prepared in the first step.
The procedure is as in step three of example 1.
IV, electrophoretic analysis
The product of step two or the product of step three was loaded on a genetic analyzer for detection (loading amount 1. mu.L). By using
STR typing was carried out by ID-X analysis software (V3.2) with the RFU value of the fluorescence peak set at 50 or more.
The results for sample No. 9 are shown in fig. 13 and 14. The detection results after conventional PCR amplification for the No. 9 exfoliated cell test material showed no detection (fig. 13), but mixed STR peak profiles were obtained after ddPCR amplification and suggested that there might be more than three mixes (fig. 14). Through comparison, the obtained mixed peak spectrum contains the DNA typing of a known victim. The source of the other donor DNA is unknown.
The results for sample No. 10 are shown in fig. 15 and 16. A single source incomplete STR peak profile was obtained after conventional PCR amplification of sample No. 10 (fig. 15), but after ddPCR amplification suggested a two-person pooled sample (fig. 16) and conditioned for preliminary resolution.
The results for sample No. 12 are shown in fig. 17 and 18. Sample No. 12 showed mixing after both conventional PCR amplification and ddPCR amplification, but mixed-map quality ddPCR outperformed traditional PCR results.
Claims (10)
1. A method of STR typing a biological sample comprising the steps of:
extracting genome DNA of a biological sample, carrying out micro-droplet digital PCR amplification by using the genome DNA as a template, sequencing an amplification product, and carrying out STR typing according to a sequencing result.
2. A method of STR typing a DNA sample, comprising the steps of:
and (3) carrying out micro-droplet digital PCR amplification by taking the DNA sample as a template, sequencing the amplified product, and carrying out STR typing according to a sequencing result.
3. A system for STR typing of a biological sample, comprising: comprises a component A for extracting DNA, a component B for carrying out micro-drop digital PCR amplification and a component C for sequencing.
4. A system for STR typing of a DNA sample, comprising: comprises a component B for carrying out the micro-drop digital PCR amplification and a component C for carrying out the sequencing.
5. Use of module A, module B and module C as claimed in claim 4 for the preparation of a system for STR typing of biological samples.
6. Use of module b and module c according to claim 5 for preparing a system for STR typing of DNA samples.
7. Use of a module for performing a microdroplet digital PCR amplification for STR typing of a biological sample.
8. Use of a module for performing digital PCR amplification in microdroplet in STR typing of a DNA sample.
9. Use of a module for performing digital PCR amplification in microdroplet in the preparation of a system for STR typing of a biological sample.
10. Use of a module for performing digital PCR amplification in the manufacture of a system for STR typing of a DNA sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN113160892A (en) * | 2021-05-25 | 2021-07-23 | 北京众诚天合***集成科技有限公司 | Method and system for determining genetic relationship of mixed DNA typing |
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