CN111718984A - A method for STR typing of forensic pooled DNA samples - Google Patents
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Abstract
本发明公开了一种对法医混合DNA样本进行STR分型的方法。本发明提供了一种对生物样本进行STR分型的方法，包括如下步骤：提取生物样本的基因组DNA，将基因组DNA作为模板，进行微滴式数字PCR扩增，将扩增产物进行测序，根据测序结果进行STR分型。所述生物样本为混合生物样本。本发明还保护一种对DNA样本进行STR分型的方法，包括如下步骤：将DNA样本作为模板，进行微滴式数字PCR扩增，将扩增产物进行测序，根据测序结果进行STR分型。所述DNA样本为混合DNA样本。本发明在法医DNA检验中有巨大的应用前景。The invention discloses a method for STR typing of forensic mixed DNA samples. The invention provides a method for STR typing of biological samples, comprising the following steps: extracting genomic DNA of biological samples, using the genomic DNA as a template, performing droplet digital PCR amplification, sequencing the amplification products, Sequencing results were used for STR typing. The biological sample is a mixed biological sample. The present invention also protects a method for STR typing on a DNA sample, comprising the following steps: using the DNA sample as a template, performing droplet digital PCR amplification, sequencing the amplified product, and performing STR typing according to the sequencing result. The DNA sample is a mixed DNA sample. The invention has a huge application prospect in forensic DNA testing.
Description
技术领域technical field
本发明涉及一种对法医混合DNA样本进行STR分型的方法。The present invention relates to a method for STR typing of forensic mixed DNA samples.
背景技术Background technique
随着DNA检验水平的提高，微量接触类检材逐渐成为DNA实验室的主要检材，而在此类检材上获得的绝大部分都是混合DNA分型(比如受害人与嫌疑人的DNA混合，2个或2个以上嫌疑人样本的混合，或者是嫌疑人与另一无关个体的混合等等)。而恰恰是这些混合样本对提供案件线索、确立侦查范围和提供法庭证据等方面有很重要的现实意义。然而，混合样本DNA图谱因混合的人数不同、混合比例不同，而造成STR峰谱多、带型复杂、峰型不稳定，导致混合样本结果分析困难、定性风险高，使得混合结果极易被舍弃或忽略，造成遗漏重要的案件信息或者使得DNA证据效力大打折扣。With the improvement of DNA testing level, trace contact samples have gradually become the main samples of DNA laboratories, and most of the samples obtained from such samples are mixed DNA typing (such as the DNA of victims and suspects). Mixing, mixing of 2 or more samples of suspects, or mixing of suspects with another unrelated individual, etc.). It is precisely these mixed samples that have important practical significance for providing clues to cases, establishing the scope of investigation and providing court evidence. However, the DNA profiles of mixed samples are different due to the number of people mixed and the mixing ratios, resulting in many STR peak spectra, complex band patterns, and unstable peak patterns, which make the analysis of mixed sample results difficult and high qualitative risk, making the mixed results easily discarded. Or ignore it, resulting in the omission of important case information or greatly reducing the effectiveness of DNA evidence.
目前国内外法医界对于混合DNA样本的检验和分析方法主要有将混合DNA图谱进行基于计算机软件的拆分，或者专家人工拆分，或者将混合DNA样品进行单细胞分离，从而获得单一DNA分型，从而从混合样本中分解出有价值的DNA信息。其中单细胞分离技术有赖于专业的显微操作设备和训练有素的操作人员，并且能够进行单细胞分离的条件受限。而基于计算机技术或者基于有经验专家的人工拆分，都依赖于高质量的混合图谱的获得，而无法改变检材中DNA混合的初始状态。但是在实际案件中，检材本身可能的混合情况是受害人DNA背景强，而嫌疑人的DNA含量相对很低，低含量DNA模板被淹没于高含量的DNA模板的海洋中。At present, the inspection and analysis methods of mixed DNA samples in the forensic circles at home and abroad mainly include the splitting of mixed DNA profiles based on computer software, manual splitting by experts, or single-cell separation of mixed DNA samples, so as to obtain single DNA typing , thereby decomposing valuable DNA information from mixed samples. Among them, single-cell isolation technology relies on professional micromanipulation equipment and well-trained operators, and the conditions for single-cell isolation are limited. However, based on computer technology or manual separation based on experienced experts, both rely on the acquisition of high-quality mixed maps, and cannot change the initial state of DNA mixing in the sample. However, in actual cases, the possible mixture of the samples themselves is that the victim's DNA background is strong, while the suspect's DNA content is relatively low, and the low-content DNA templates are submerged in the ocean of high-content DNA templates.
微滴式数字PCR技术是将样本通过微滴发生器进行微滴化处理，利用油包水技术将其“分割”为2×104个1nL体积的微滴后进行PCR反应，每一个微滴都是一个独立的PCR反应体系，该方法通量高，操作简单、成本低。The droplet digital PCR technology is to process the sample into droplets through a droplet generator, and use the water-in-oil technology to "divide" it into 2×10 4 droplets of 1nL volume, and then carry out the PCR reaction. All are an independent PCR reaction system, the method has high throughput, simple operation and low cost.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种对法医混合DNA样本进行STR分型的方法。The purpose of the present invention is to provide a method for STR typing of forensic mixed DNA samples.
本发明提供了一种对生物样本进行STR分型的方法，包括如下步骤：提取生物样本的基因组DNA，将基因组DNA作为模板，进行微滴式数字PCR扩增，将扩增产物进行测序，根据测序结果进行STR分型。所述生物样本为混合生物样本，例如来源于2个以上个体的混合血样，来源于2个以上个体的混合细胞样等。所述方法用于法医鉴定领域。所述微滴式数字PCR扩增的反应程序为：95℃1min；94℃30s、59℃1.5min，29个循环；60℃10min；98℃10min。所述微滴式数字PCR扩增的升降温速度设置为2℃/s。所述微滴式数字PCR扩增的反应体系(20μL)：2μL模板、2μL 
本发明还保护一种对DNA样本进行STR分型的方法，包括如下步骤：将DNA样本作为模板，进行微滴式数字PCR扩增，将扩增产物进行测序，根据测序结果进行STR分型。所述DNA样本为混合DNA样本，即来源于2个以上个体的DNA样本，例如来源于2个以上个体的混合血样的DNA样本，来源于2个以上个体的混合细胞样的DNA样本等。所述方法用于法医鉴定领域。所述微滴式数字PCR扩增的反应程序为：95℃1min；94℃30s、59℃1.5min，29个循环；60℃10min；98℃10min。所述微滴式数字PCR扩增的升降温速度设置为2℃/s。所述微滴式数字PCR扩增的反应体系(20μL)：2μL模板、2μL 
本发明还保护一种对生物样本进行STR分型的系统，其特征在于：包括用于进行DNA提取的组件甲、用于进行微滴式数字PCR扩增的组件乙和用于进行测序的组件丙。所述组件乙为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护一种对DNA样本进行STR分型的系统，其特征在于：包括用于进行微滴式数字PCR扩增的组件乙和用于进行测序的组件丙。所述组件乙为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行DNA提取的组件甲、用于进行微滴式数字PCR扩增的组件乙和用于进行测序的组件丙在制备对生物样本进行STR分型的系统中的应用。所述组件乙为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行微滴式数字PCR扩增的组件乙和用于进行测序的组件丙在制备对DNA样本进行STR分型的系统中的应用。所述组件乙为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行微滴式数字PCR扩增的组件在对生物样本进行STR分型中的应用。所述STR分型为法医鉴定领域的STR分型。所述用于进行微滴式数字PCR扩增的组件为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行微滴式数字PCR扩增的组件在对DNA样本进行STR分型中的应用。所述STR分型为法医鉴定领域的STR分型。所述用于进行微滴式数字PCR扩增的组件为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行微滴式数字PCR扩增的组件在制备对生物样本进行STR分型的系统中的应用。所述STR分型为法医鉴定领域的STR分型。所述用于进行微滴式数字PCR扩增的组件为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
本发明还保护用于进行微滴式数字PCR扩增的组件在制备对DNA样本进行STR分型的系统中的应用。所述STR分型为法医鉴定领域的STR分型。所述用于进行微滴式数字PCR扩增的组件为微滴生成仪、梯度PCR扩增仪、ddPCR Super Mix for probes(no dUTP)、
以上任一所述遗传分析仪为Applied Biosystems公司生产的3500XL型遗传分析仪。Any one of the genetic analyzers described above is a 3500XL genetic analyzer produced by Applied Biosystems.
以上任一所述梯度PCR扩增仪为美国Bio-Rad公司生产的T100型梯度PCR仪。Any one of the above gradient PCR amplifiers is a T100 type gradient PCR instrument produced by Bio-Rad Company in the United States.
将9947A、007等DNA标准物质制成不同混合比例的DNA混合样本并搜集实际案件中混合DNA检材，分别使用传统PCR扩增和微滴式数字PCR扩增，扩增产物经遗传分析仪检测和STR图谱分析。经过比较两种方法的扩增效果，微滴式数字PCR对混合DNA样本扩增结果更好。在9947A与007混合比例低至20:1时仍可较好的检出007的STR峰谱，在实际案件的混合DNA样本中可以获得质量更好的DNA混合图谱。微滴式数字PCR技术可以提高混合DNA中低浓度组分的扩增效力，削弱扩增偏嗜效应，有利于DNA混合图谱的判读，在法医混合DNA检验中有很好的应用潜力。DNA standard materials such as 9947A and 007 were made into DNA mixed samples with different mixing ratios, and the mixed DNA samples in actual cases were collected. Traditional PCR amplification and droplet digital PCR amplification were used respectively, and the amplified products were detected by genetic analyzer. and STR profile analysis. After comparing the amplification effects of the two methods, droplet digital PCR has better amplification results for mixed DNA samples. When the mixing ratio of 9947A and 007 is as low as 20:1, the STR peak spectrum of 007 can still be detected well, and a better quality DNA mixed spectrum can be obtained in the mixed DNA samples of actual cases. Droplet digital PCR technology can improve the amplification efficiency of low-concentration components in mixed DNA, weaken the amplification bias effect, and is conducive to the interpretation of DNA mixed maps. It has good application potential in forensic mixed DNA testing.
到目前为止，还没有将ddPCR技术应用到法医学领域混合DNA扩增和分析上，是一项技术空白。本发明利用微滴式数字PCR油包水可以生成2万个微滴的特点，改变反应体系和调整反应参数，削弱DNA的扩增偏嗜作用，可以提高混合样本中低组分DNA扩增概率，调整DNA产物混合比例，更加利于DNA图谱分析，深度挖掘混合DNA样本信息，为混合DNA检验提供新思路和新方法。本发明提供的方法，使得低含量的DNA模板能得到均等的扩增机会，具有更高的扩增灵敏度和精确度，能够检测到混合比例大于5％(20:1)的低组分混合物。本发明在法医DNA检验中有巨大的应用前景。So far, the ddPCR technology has not been applied to the mixed DNA amplification and analysis in the field of forensic medicine, which is a technical blank. The present invention utilizes the characteristic that microdroplet digital PCR water-in-oil can generate 20,000 microdroplets, changes the reaction system and adjusts the reaction parameters, weakens the amplification bias of DNA, and can improve the amplification probability of low-component DNA in mixed samples , adjust the mixing ratio of DNA products, which is more conducive to the analysis of DNA profiles, deeply mine the information of mixed DNA samples, and provide new ideas and new methods for mixed DNA testing. The method provided by the present invention enables low-content DNA templates to obtain equal amplification opportunities, has higher amplification sensitivity and accuracy, and can detect low-component mixtures with a mixing ratio greater than 5% (20:1). The invention has a huge application prospect in forensic DNA testing.
附图说明Description of drawings
图1为9947:007＝1:1时常规PCR扩增结果。Figure 1 shows the results of conventional PCR amplification when 9947:007=1:1.
图2为9947:007＝2:1时常规PCR扩增结果。Figure 2 shows the results of conventional PCR amplification when 9947:007=2:1.
图3为9947:007＝5:1时常规PCR扩增结果。Figure 3 shows the results of conventional PCR amplification when 9947:007=5:1.
图4为9947:007＝10:1时常规PCR扩增结果。Figure 4 shows the results of conventional PCR amplification when 9947:007=10:1.
图5为9947:007＝20:1时常规PCR扩增结果。Figure 5 shows the results of conventional PCR amplification when 9947:007=20:1.
图6为9947:007＝50:1时常规PCR扩增结果。Figure 6 shows the results of conventional PCR amplification when 9947:007=50:1.
图7为9947:007＝1:1时ddPCR扩增结果。Figure 7 shows the results of ddPCR amplification when 9947:007=1:1.
图8为9947:007＝2:1时ddPCR扩增结果。Figure 8 shows the results of ddPCR amplification when 9947:007=2:1.
图9为9947:007＝5:1时ddPCR扩增结果。Figure 9 shows the ddPCR amplification results when 9947:007=5:1.
图10为9947:007＝10:1时ddPCR扩增结果。Figure 10 shows the results of ddPCR amplification when 9947:007=10:1.
图11为9947:007＝20:1时ddPCR扩增结果。Figure 11 shows the ddPCR amplification results when 9947:007=20:1.
图12为9947:007＝50:1时ddPCR扩增结果。Figure 12 shows the ddPCR amplification results when 9947:007=50:1.
图13为9号样品常规扩增结果。Figure 13 shows the routine amplification results of sample No. 9.
图14为9号样品ddPCR扩增结果。Figure 14 shows the ddPCR amplification results of sample No. 9.
图15为10号样品常规扩增结果。Figure 15 shows the routine amplification results of sample No. 10.
图16为10号样品ddPCR扩增结果。Figure 16 shows the ddPCR amplification results of sample No. 10.
图17为12号样品常规扩增结果。Figure 17 shows the routine amplification results of sample No. 12.
图18为12号样品ddPCR扩增结果。Figure 18 shows the ddPCR amplification results of sample No. 12.
具体实施方式Detailed ways
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.
STR(short tandem repeat，短片段重复序列)广泛存在于人类及哺乳动物的基因组中，具有高度多态性，它们一般由2-6个核苷酸构成一个核心序列，核心序列串联重复排列，由核心序列重复数目的变化产生长度多态性。对于一个特定的个体，染色体上某个特定位置的重复序列的重复次数是固定的，而对于不同的个体在同一位置处的重复次数可能不同，这就构成了人群中这些重复序列的多态性。STRs (short tandem repeats) are widely present in the genomes of humans and mammals and are highly polymorphic. They generally consist of 2-6 nucleotides to form a core sequence. The core sequence is repeated in tandem, consisting of Variation in the number of core sequence repeats produces length polymorphisms. For a specific individual, the number of repetitions of a repeat sequence at a specific position on the chromosome is fixed, while the number of repeats at the same position may be different for different individuals, which constitutes the polymorphism of these repeat sequences in the population .
9947A人类基因组DNA标准物(简称9947A标准物)，女性，1ng/μl，为上海博升生物科技有限公司产品，产品目录号为AM100035，产品链接为http://www.biosun.cn/news/7718.htm。9947A human genomic DNA standard (referred to as 9947A standard), female, 1ng/μl, is a product of Shanghai Bosheng Biotechnology Co., Ltd., the product catalog number is AM100035, and the product link is http://www.biosun.cn/news/ 7718.htm.
007人类基因组DNA标准物(简称007标准物)，男性，0.1ng/μl，为Thermo公司的
AmpFLSTRTMIdentifilerTMPCR Amplification Kit，为Thermo公司产品，产品目录号为4322288，产品链接为AmpFLSTR TM Identifiler TM PCR Amplification Kit, a product of Thermo, catalog number 4322288, the product link is
https://www.thermofisher.com/order/catalog/product/4322288？SID＝srch-srp-4322288。https://www.thermofisher.com/order/catalog/product/4322288? SID=srch-srp-4322288.
Bio-Rad QX200微滴式数字PCR系统，为美国Bio-Rad公司产品，由QX200微滴生成仪和QX200微滴分析仪组成，实施例中仅用到微滴生成仪。梯度PCR扩增仪为美国Bio-Rad公司生产的T100型梯度PCR仪。遗传分析仪为Applied Biosystems公司生产的3500XL型遗传分析仪，
ddPCR Super Mix for probes(no dUTP)，为BIO-RAD公司产品，产品目录号为1863023，产品链接为ddPCR Super Mix for probes (no dUTP) is a product of BIO-RAD Company, the catalog number is 1863023, and the product link is
http://www.bio-rad.com/en-us/sku/1863023-ddpcr-supermix-for-probes-no-dutp？ID＝1863023。http://www.bio-rad.com/en-us/sku/1863023-ddpcr-supermix-for-probes-no-dutp?f ID=1863023.
采用
表1Table 1
实施例1、检测标准物混合样本Example 1, detection standard mixed sample
一、混合样本的制备1. Preparation of mixed samples
6个混合样本的配比见表2。The proportions of the 6 mixed samples are shown in Table 2.
表2Table 2
二、常规PCR扩增2. Conventional PCR amplification
采用
反应体系为20μL，由2μL模板、2μL 
模板分别为步骤一制备的6个混合样本。The templates are the 6 mixed samples prepared in step 1, respectively.
反应程序见试剂盒说明书，29个循环。The reaction procedure is shown in the kit instructions, 29 cycles.
三、微滴式数字PCR扩增3. Droplet Digital PCR Amplification
反应体系为20μL，由2μL模板、2μL 
模板分别为步骤一制备的6个混合样本。The templates are the 6 mixed samples prepared in step 1, respectively.
1、分别将20μL反应体系和70μL微滴生成油加在微滴发生卡上，盖上胶垫后放入微滴生成仪中，生成微滴。1. Add 20μL of the reaction system and 70μL of droplet generation oil to the droplet generation card, cover with a rubber pad, and put them into the droplet generator to generate droplets.
2、将步骤1得到的约40μL微滴转移到96孔板中，用热封膜仪进行封膜，然后置于T100型梯度PCR扩增仪中进行反应。反应程序：95℃1min；94℃30s、59℃1.5min，29个循环；60℃10min；98℃10min；15℃∞；升降温速度设置为2℃/s。产物4℃保存。2. Transfer about 40 μL of microdroplets obtained in step 1 to a 96-well plate, seal the film with a heat sealer, and then place it in a T100 gradient PCR amplifier for reaction. Reaction program: 95 °C for 1 min; 94 °C for 30 s, 59 °C for 1.5 min, 29 cycles; 60 °C for 10 min; 98 °C for 10 min; 15 °C ∞; the ramp rate was set to 2 °C/s. The product was stored at 4°C.
3、将步骤2的产物离心，初步去掉管底的油相，加10μL TE溶液，在通风橱中加入40μL氯仿，上下混匀后转入一个1.5ml带盖离心管中，最高速度涡旋震荡1分钟，15000g离心10分钟，弃去上层氯仿相，转移至一个新的1.5ml离心管中。3. Centrifuge the product of step 2, preliminarily remove the oil phase at the bottom of the tube, add 10 μL of TE solution, add 40 μL of chloroform in a fume hood, mix up and down, transfer to a 1.5 ml centrifuge tube with a lid, and vortex at the highest speed. Centrifuge at 15000g for 1 min for 10 min, discard the upper chloroform phase and transfer to a new 1.5 ml centrifuge tube.
四、电泳分析4. Electrophoresis analysis
步骤二的产物或步骤三的产物上样于遗传分析仪进行检测(上样量为1μL)。用
6个混合样本常规PCR后电泳分析的结果见图1至图6。当混合样品中女性成分与男性成分比例为5：1时，还可以勉强检出混合STR峰谱，并且基本能够对混合样本进行人工拆分(图3)，但当混合样品中女性成分与男性成分比例变为10：1时，可以从性别位点Amel检出有极低的Y峰，但是其他基因座上想拆分出007的等位基因已经很困难(图4)，而当混合比例提高到20:1甚至50:1时，从STR峰谱判读已经只剩下9947的单一分型，较难发现混合成分了(图5，图6)。The results of electrophoresis analysis of 6 mixed samples after conventional PCR are shown in Figures 1 to 6. When the ratio of female components to male components in the mixed sample is 5:1, the mixed STR peak spectrum can be barely detected, and the mixed samples can be divided manually (Figure 3). When the component ratio becomes 10:1, a very low Y peak can be detected from the sex locus Amel, but it is difficult to split the 007 allele at other loci (Figure 4). When it is increased to 20:1 or even 50:1, only 9947 single types are left from the interpretation of the STR peak spectrum, and it is difficult to find mixed components (Figure 5, Figure 6).
6个混合样本微滴式数字PCR扩增后电泳分析的结果见图7至图12。在男女混合比例为1:1和1:2时(图7，图8)，与常规PCR的效果差异不明显，但当混合比例提高到5:1时，经微滴式数字PCR扩增的结果明显好于传统PCR，可以清晰的看出男性成分的混合，并且男性的STR位点可以非常明确的拆分而获得单一分型(图9)，当混合比例为10:1时，混合图谱依然可以对男性成分进行确认和拆分(图10)。即使当混合比例提高至20:1和50:1，仍然可以在Amel位点检出男性成分，提示该样品为混合样品，可以继续做Y-STR分型，而不会造成混合图谱的信息遗漏或者误判，在部分基因座上仍然可以获得男性成分的STR分型(图11，图12)。可见改用微滴式数字PCR扩增，混合图谱质量有了明显提高。Figures 7 to 12 show the results of electrophoresis analysis of 6 mixed samples after droplet digital PCR amplification. When the male-female mixing ratio was 1:1 and 1:2 (Fig. 7, Fig. 8), the effect of conventional PCR was not significantly different, but when the mixing ratio was increased to 5:1, the PCR amplified by droplet type PCR The results are significantly better than those of traditional PCR, and the mixture of male components can be clearly seen, and the STR loci of males can be very clearly split to obtain a single type (Figure 9). When the mixing ratio is 10:1, the mixed map The male component can still be identified and split (Figure 10). Even when the mixing ratio is increased to 20:1 and 50:1, male components can still be detected at the Amel site, indicating that the sample is a mixed sample, and Y-STR typing can be continued without causing information omission of the mixed map Or misjudgment, STR typing of male components can still be obtained at some loci (Fig. 11, Fig. 12). It can be seen that the use of droplet digital PCR amplification has significantly improved the quality of the mixed map.
实施例2、检测实际样本Example 2. Detection of actual samples
供试样本：来自中心日常接检的血样混合物(12号样品)、脱落细胞混合物(9号样本、10号样本)。Samples: blood sample mixture (sample No. 12) and exfoliated cell mixture (sample No. 9, sample No. 10) from the center's daily inspection.
一、分别提取各个供试样本的基因组DNA。1. Extract the genomic DNA of each sample.
二、常规PCR扩增2. Conventional PCR amplification
采用AmpFLSTRTMIdentifilerTMPCR Amplification Kit并按说明书操作。Use AmpFLSTR ™ Identifiler ™ PCR Amplification Kit and follow the instructions.
每个反应体系中，基因组DNA的含量为0.5ng。In each reaction system, the content of genomic DNA was 0.5 ng.
模板分别为步骤一制备的各个供试样本的基因组DNA。The templates are the genomic DNA of each sample prepared in step 1, respectively.
三、微滴式数字PCR扩增3. Droplet Digital PCR Amplification
反应体系为20μL，由2μL模板(2微升模板中的DNA含量为0.5ng)、2μL
模板分别为步骤一制备的各个供试样本的基因组DNA。The templates are the genomic DNA of each sample prepared in step 1, respectively.
方法同实施例1的步骤三。The method is the same as step 3 of Example 1.
四、电泳分析4. Electrophoresis analysis
步骤二的产物或步骤三的产物上样于遗传分析仪进行检测(上样量为1μL)。用
9号样本的结果见图13和图14。对于9号脱落细胞检材在常规PCR扩增后的检测结果显示为未检出(图13)，但是经过ddPCR扩增后获得混合STR峰谱并且提示可能为三人以上混合(图14)。经过比对，获得的混合峰谱中包含一个已知受害人的DNA分型。其他供体DNA来源未知。The results for sample No. 9 are shown in Figures 13 and 14. The detection result of No. 9 exfoliated cell sample after conventional PCR amplification showed that it was not detected (Figure 13), but a mixed STR peak spectrum was obtained after ddPCR amplification and suggested that more than three people may be mixed (Figure 14). After alignment, the resulting mixed peak spectrum contains a DNA type of a known victim. The source of the other donor DNA is unknown.
10号样本的结果见图15和图16。10号样品的常规PCR扩增后获得一个单一来源的不完整STR峰谱(图15)，但是经ddPCR扩增后提示为两人混合样本(图16)并且有条件进行初步拆分。The results of sample No. 10 are shown in Figure 15 and Figure 16. After routine PCR amplification of sample No. 10, an incomplete STR peak spectrum from a single source was obtained (Figure 15), but after ddPCR amplification, it was suggested that it was a mixed sample of two people (Figure 15). 16) and conditionally conduct preliminary splitting.
12号样本的结果见图17和图18。12号样本经常规PCR扩增和ddPCR扩增后都显示为有混合，但是混合图谱质量ddPCR优于传统PCR结果。The results of sample No. 12 are shown in Figure 17 and Figure 18. Sample No. 12 showed mixing after conventional PCR amplification and ddPCR amplification, but the mixed map quality ddPCR was better than the traditional PCR results.
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