接触DNA检材提取纯化方法的比较及法医学应用
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摘要
接触DNA (touch DNA)检验是法医DNA检验的难点和热点之一。法医鉴定中的接触DNA检材,是指含有人体皮肤粘膜的脱落细胞的检材,包括含口腔脱落细胞的检材、含体表脱落细胞的检材、人体排泄物或分泌物等。凡是与人体皮肤粘膜接触过的物品,都可能留下接触者的脱落细胞。这些留有脱落细胞的物证可以存在于犯罪现场,也可存在于犯罪嫌疑人及被害对象的衣物、用具等客体上。随着犯罪分子反侦察手段的日益狡猾,明显的生物物证(精斑、血斑、毛发、人体组织等)常被有目的地销毁和破坏,而来自人体的代谢脱落细胞由于微小痕量,常被忽略,如遗留在烟蒂、饮料罐、果核、口香糖上的口腔脱落上皮细胞,遗留在手套、衣物、帽子、指纹上的皮肤脱落上皮细胞,等等。对于这些潜在的微量生物物证,一旦成功地获得了关键物证的DNA分型,往往就能够成为案件侦破的关键线索,并为案件定性及法庭量刑提供科学证据。
如何从微量接触检材中提取到足够的DNA以满足法医学检验的需要,选择合适的提取方法非常关键,是法医物证检验工作者最关注的难题之一。接触DNA检材与常见的生物检材如血痕、精斑相比,属于疑难生物检材。首先,肉眼观察很难判断接触细胞在载体上的具体部位,盲目提取难以获得所需靶细胞；其次,擦拭面积太大会降低靶DNA的浓度,并带来外源物质的污染和干扰；第三,接触DNA检材含有的DNA往往是微量的,属于低拷贝数目(Low copy number,LCN)模板,即“DNA含量少于l00pg,相当于15个双倍体或30个单倍体细胞的生物样本”。因此接触DNA检验不同于常规生物检材的DNA检验,更需系统研究。
目前关于接触DNA提取纯化方法的文献很多,概括起来有Chelex法、磁珠法、有机溶剂法(有机法)、过柱法、硅珠法、硅胶膜法等。但多为针对某种方法对某种类型接触DNA的提取或案例报道,对不同提取纯化方法获得的现场不同类型的接触DNA的质与量的横向比较缺乏系统研究。本研究采用Quantifiler人类DNA定量试剂盒(AB, USA)实时定量技术对目前常用的提取纯化方法包括Chelex法、磁珠提取法(DNA IQ磁珠法、EQ国产磁珠法)、有机溶剂提取法、Microcon过柱法提取的接触DNA进行定量,同时用Identifiler或Sinofiler复合扩增系统(AB,USA)扩增,在AB 3130遗传分析仪上进行STR (short tandem repeat,短串联重复)分型。综合DNA定量和STR分型结果,对各种提取纯化方法获得的DNA的质和量进行评估和系统比较,以建立有效的接触DNA提取纯化方法。
1. Chelex法提取接触DNA的实时定量研究
对使用后放置1个月以内的30个饮料瓶口或吸管、30个矿泉水瓶(杯)口分别采用不洗涤直接加Chelex-100提取与洗涤+Chelex-100的方法提取DNA,通过PCR定量和STR检测,比较2种处理方式获得的DNA平均含量、IPC Ct值、STR检验成功率；对使用后放置1个月以内的10个新鲜饮料瓶口或吸管,使用后分别放置1个月和放置2个月的20把牙刷、20枚烟蒂,分别采用洗涤+Chelex-100和洗涤+Chelex-100+蛋白酶K(PK)的方法提取DNA,通过PCR定量和STR检测,比较2种处理方式获得的DNA平均含量、IPC Ct值、STR检验成功率。研究结果表明,Chelex-100提取前加浸泡洗涤的步骤,可去除部分可溶性杂质,有利于提高获得的DNA量和STR检验成功率,同时降低反映PCR抑制物存在的IPC Ct值。室温放置1个月左右的新鲜接触DNA检材用Chelex法提取时,是否加蛋白酶K对提取的DNA量和STR检验成功率无显著影响；室温放置2个月以上的接触DNA检材用Chelex法提取时,加蛋白酶K消化后获得的DNA量和STR检验成功率均高于不加蛋白酶K的样品,差异有显著性意义。本研究对180份10种实际案件的接触DNA检材用Chelex法提取,烟蒂获得的平均DNA含量最高达175.85 ng,剃须刀较低为7.80 ng。除烟蒂、口香糖采用常规体系提取外,其它检材采用小体系提取,平均DNA浓度均在0.2ng/μl以上,采用2μll模板复合扩增,除衣服、剃须刀的STR分型成功率较低为50%外,其它检材的STR分型成功率均在60%以上。
2.磁珠法提取接触DNA的实时定量研究
对烟蒂、牙刷、手套等3种接触DNA检材各10个分别采用95℃裂解液直接裂解、70℃裂解液直接裂解和预消化(TNE+SDS+PK)等3种前处理方式后用DNA IQ磁珠(Promega, USA)提取DNA,测定并比较三种前处理方式后用DNAIQ磁珠法提取获得的DNA平均含量和IPCCt值。研究结果表明,接触DNA检材采用先加TNE、SDS、PK等消化,然后再用磁珠纯化的方法获得的DNA量高于单纯用裂解液裂解的方法提取的DNA量,有助于提高微量和污染接触DNA检材的STR检验成功率。但该处理方法操作相对繁琐,耗时长,除了污染严重的微量接触DNA检材外,大部分接触DNA检材单纯采用直接裂解法就可以获得足以进行STR分析的DNA。裂解温度方面,95℃裂解与70℃裂解的DNA获得量无显著性差异。对151份8种实际案件的接触DNA检材采用95℃直接裂解的DNAIQ磁珠法在MaxwellTM 16自动仪上提取DNA。结果表明除果核平均DNA获得量为9.51ng以外,其它7种接触检材的平均DNA获得量均大于10ng。采用30μl的小洗脱体积,平均DNA浓度可达0.3ng/μl,采用8μl体系3μl模板扩增,检验成功率最低的果核也达60%。EQ国产磁珠法与DNA IQ磁珠法相比,不仅价格便宜,还少了1个裂解液洗涤和1个洗涤液洗涤的步骤,操作更为简单,但对接触DNA的提取效率无显著性差异。对138份6种实际案件的接触DNA检材在自动化工作站经EQ国产磁珠法提取DNA,采用8μl体系3μl模板扩增,除对饮料瓶口的检验成功率较低,为60%外,对烟蒂、口香糖、手套等接触DNA检材的检验成功率也能达到70%以上。因此也适合接触DNA的提取。
3.5种接触DNA检材提取纯化方法的比较
将稀释为lOng、100ng的标准品DNA,分别采用Chelex法、DNA IQ磁珠法、EQ国产磁珠法、有机法、Microcon过柱法等5种DNA提取纯化方法处理,然后进行PCR定量,比较5种DNA提取纯化方法对标准品DNA的回收率。对烟蒂和牙刷2种常见的接触DNA检材分别采用Chelex法、95℃直接裂解的DNA IQ磁珠法和EQ国产磁珠法提取DNA,通过PCR定量和STR检测分析,比较3种提取方法提取的DNA平均含量、IPC Ct值和STR检验效果；对30例污染严重的接触DNA检材,先用TNE、SDS、PK消化,然后将消化液平均分成2份,1份用DNA IQ磁珠法纯化,1份用有机法抽提,通过PCR定量和STR检测分析,比较磁珠法与有机法对接触DNA的纯化效果；将Chelex法提取的15例5种接触DNA溶液用Microcon100超滤柱进行纯化浓缩,通过PCR定量和STR检测分析,比较Microcon过柱法浓缩前后的浓度、体积、IP Ct值及回收率。结果表明,Chelex法对DNA的提取无损失,磁珠法、有机法、Microcon过柱法对DNA的提取纯化均有不同程度的损失。5种提取纯化方法对DNA的回收率从高到低大致为Chelex法>Microcon过柱法>DNA IQ磁珠法>EQ国产磁珠法>有机法。Chelex法提取DNA,操作简单快速,整个提取过程均在一个离心管中进行,避免了DNA在提取过程中的损耗,对污染轻、杂质少的接触DNA检材,用Chelex法提取最为方便快捷。但Chelex法不能去除DNA溶液中的杂质和降解的DNA碎片,因此,Chelex法提取的陈旧接触检材的DNA进行STR扩增时,即使模板量在试剂盒推荐的范围内,也容易出现等位基因扩增不平衡的现象。磁珠法与Chelex法相比,虽然提取过程中会造成DNA的损失,但提取的DNA纯度高,能有效去除100bp以下的DNA小片段和样品中的色素、蛋白质等杂质,因此,STR扩增时峰高比较均衡,等位基因扩增不平衡的现象不明显。磁珠法与有机法对接触DNA的回收量和IPC Ct值虽然无显著性差异,但磁珠法避免了有机溶剂对操作者的潜在危害,操作简单,耗时短,因此,更适合微量、污染检材的DNA提取及自动化操作。Microcon过柱法操作简单,但对接触DNA的回收效果不一,100μl的起始体积,过柱浓缩后的体积在8-25μl之间,平均为15μl。过柱后,DNA浓度平均约增加5倍,对DNA的回收率平均为61%,但反映PCR抑制物存在的IPC Ct值无显著降低。同时,Microcon过柱法的成本也高于磁珠法。因此,Microcon过柱法对接触DNA的提取纯化应用有限,只适合污染轻的接触DNA的纯化浓缩。
本研究通过对5种常见的提取纯化方法获得的DNA的质和量进行系统比较和评估,为规范法医检验中接触DNA检材的提取送检以及法医工作者如何根据检材、实验室设备等情况选择合适有效的接触DNA提取纯化方法提供了科学依据,有利于提高接触DNA检材的检验成功率,充分发挥接触DNA检材的证据价值。
Touch DNA analysis is one of the difficulties and hotspots of forensic DNA typing. The touch DNA samples in forensic DNA typing means the objects with sloughed off skin and mucous membrane cells. The objects which contacted with human skin or mucous membrane are likely to contain exfoliated epithelial cells. The biological evidence not only exists on the crime scene but also can be found on the clothing, utensils and other objects of criminal suspects or the victims. As criminals' anti-surveillance means become increasingly strong, obvious biological evidence (such as sperm spots, blood stains, hair, human tissue, etc.) are often purposely destroyed. Small amount epithelial cells from the body are often neglected, such as the oral exfoliated epithelial cells left on cigarette butts, beverage cans, fruit-stones, chewing gums, the skin shedding epithelial cells left in the gloves, clothes, hats, fingerprints and so on. However, it could be a vital clues to determine the nature of the case and provide scientific evidence for the court, once the touch DNA from these potential minute biological evidence can be typed successfully.
To satisfy the needs of the forensic case detection, it's very important to select the appropriate DNA extraction and purification method for obtaining sufficient DNA from the touch cells which are minute. Touch DNA samples belong to difficult samples in comparison of to common biological samples such as bloodstains, semen stains. The reasons are the following:First, it is difficult to judge the exact location on the carrier and obtain target cells only by naked eyes; Second, it will decrease target DNA concentration and bring exogenous pollution and disturbance when extracted with more wiped area; Third, touch DNA samples often contain low copy number (LCN) templates, which means "DNA amount is less than 100pg, equivalent to 15 diploid or 30 haploid cell. " Therefore, touch DNA testing is different from the conventional biological samples and needs to study systemically.
Nowadays, there are numerous references on touch DNA extraction and purification methods. In general, there are the Chelex-100 method, the magnetic beads method, the organic method (phenol-chloroform method), spin column method, the silica beads method, the silica membrane method, and so on. But mostly references only aimed at certain method of DNA extraction about certain types of touch DNA or simply case reports. There are fewer horizontal comparisons of DNA quality and quantity among the different extraction and purification methods. In this study, DNA extracted and purified by the Chelex-100 method, the magnetic beads method (DNA IQ system and EQ system), organic extraction and the Microcon100 column (Millipore, USA) method was quantified by the real-time PCR with Quantifiler Human DNA Quantification kit. STR typing with IdentifierTM or SinofilerTM multiplex STR amplification system and AB 3130 genetic analyzer(AB, USA) were also used to detect the quality of extracted touch DNA. By systematic comparison between quantification and STR typing, proper experimental evidence could help the r standardization of collecting touch DNA evidence and determination on how to select an appropriate and effective method of extraction and purification.
The results are as follows:
1. Real time quantificational study of DNA extracted by Chelex-100 method from touch DNA samples
DNA was extracted from the familiar touch DNA samples(beverage cans or sucker, cigarette butts, toothbrushes) respectivel by no-washing Chelex-100 method, washing+Chelex-100 method and washing+Chelex-100+proteinase K method. The DNA average amounts, IPC Ct and success rate of STR typing were observed and compared by PCR quantification and STR analysis. The results are as follows: The steps of immersion washing in Chelex-100 extraction can remove some of the soluble inhibitor, which can help increasing DNA quality, improving STR typing and reducing the IPC Ct which could reflect the PCR inhibitor of existing. For fresh epithelial cell samples keeped at room temperature in about 1 month, there was no significant impact on DNA yield and STR typing by Chelex method, whether plus proteinase K or not. But for more than 2 months of epithelial cell samples, it would increase more DNA yield and STR typing with proteinase K addition in Chelex-100 extraction than without. The difference was significant. In 180 cases of touch DNA extracted by Chelex method, the average DNA yield from cigarette butts was highest (175.85ng) and the average DNA yield from shavers was lower(7.80ng). Except cigarette butts, chewing gums and toothbrushs were extracted by standard volume chelex-100 protocol, other samples were for little volume chelex-100 method. The average DNA concentration for 180 samples was beyond 0.2ng/μl. The success rates of STR typing for clothes and shavers were 50%, and the success rates of STR typing for other touch DNA samples were over 60% with 2μl templet.
2. Real time quantificational study of DNA extracted by the magnetic beads method from touch DNA samples
DNA was extracted from the 3 touch DNA samples(10 cigarette butts,10 toothbrushes and 10 gloves) by DNA IQ system respectively after three preprocess protocols(95℃direct lysis,70℃direct lysis and digesting with TNE, SDS, and proteinase K). The DNA average amounts, IPC Ct and success rate of STR typing were observed and compared between PCR quantification and STR analysis. The results are as follows:It yields more DNA by digesting samples with TNE, SDS, and proteinase K before the magnetic beads purification than using direct lysis protocol, which could increase STR typing for minute and polluted smples. However, this preprocess protocol is relatively cumbersome and time-consuming. Except seriously polluted and minute DNA samples, most touch DNA samples could get enough DNA for STR analysis only by direct lysis protocol. As to lysis temperature, there is no significant difference between 95℃and 70℃in the quantity of DNA yield. After using direct lysis (95℃) protocol,151 cases of the eight kinds of touch DNA samples were extracted DNA in MaxwellTM 16 by DNA IQ magnetic beads methods (Promega). DNA quantification result shew that the average DNA yields were more than lOng for each touch DNA, except the DNA from fruit-stones,whose average yield was 9.51ng. Using 30μl elution volume, the average DNA concentrations could come up to 0.3ng/μl, and the success rate of STR typing from fruit-stones could reach to 60% with 3μl templet in 8μl amplification system. Compared with the DNA IQ System, the EQ magnetic beads kit made in China is not only more simple and much cheaper, but also lack of a lysis buffer washing and a wash buffer washing steps. But there is no significant difference in DNA extraction efficiency.138 cases touch DNA samples were extracted DNA in automation workstation by the EQ magnetic beads methods. Except the lower success rate of STR typing from beverage cans (60%), the success rate from cigarette butts, chewing gums, and gloves were about 70% with 3μl templet in 8μl amplification system. So the EQ magnetic beads kit is also suitable for touch DNA extraction.
3. The comparison of 5 touch DNA extraction and purification methods
The standard DNA diluted to 10ng, 100ng was treated respectively by 5 touch DNA extraction and purification methods(the Chelex-100 method, DNA IQ system, the EQ magnetic beads method, organic extraction and the Microcon100 column method). The recovery rates of standard DNA treated with 5 methods were compared by PCR quantification. DNA samples were extracted from cigarette butts and toothbrushes the 2 touch DNA samples respectively by Chelex method,95℃direct lysis DNA IQ method and the EQ magnetic beads methods. The DNA average amounts, IPC Ct and success rate of STR typing were compared by PCR quantification and STR analysis. The DNA samples were purified respectively by DNA IQ system and the organic method from the 30 polluted touch DNA samples after digesting by TNE, SDS and PK. The purification effects were compared by PCR quantification and STR analysis. The 15 touch DNA samples treated by the Chelex method were purified and concentrated by Microcon 100. The purification and concentration effects were compared by PCR quantification and STR analysis. The results are as follows:In general, there isn't any loss of DNA with the Chelex method to extract it. However, other protocols of DNA extraction and purification, such as the magnetic beads method, the organic method and the Microcon 100 column method, would lose some DNA. Comparatively, the rate of DNA recovery by those methods can be ranked as:Chelex> Microcon> DNA IQ System> EQ magnetic beads method (made in China)>Organic extraction. The Chelex method is simple and fast, the process of which is carried out in a centrifuge tube so that the loss of the DNA in extraction process was avoided. So the Chelex method is preferred to extract touch DNA samples with less pollution and low impurity. The magnetic beads method could result in the loss of the DNA, but yield DNA of high purity. It could concentrate DNA by adjusting elution volume and it is suitable for automatic operations. Organic extraction method is the classic method of DNA extraction. Organic method may obtain 200bp-1000kb genome DNA, the purity of which is high. DNA was extracted from 30 cases of polluted touch DNA samples by DNA IQ System and the organic method, there was no significant difference of the DNA recovery rate and IPC Ct values of DNA extracred by DNA IQ System and the organic method. But the magnetic beads method could avoid the potential hazard of operator caused by organic solvents, more simple operation and timesaving. In conclusion, the magnetic beads method is more suitable for minute and polluted samples of DNA extraction and automation. So the magnetic beads method become the first choice for minute and polluted DNA samples. The operations of Microcon100 column method is simple, but it could not filtered macromolecular impurities(such as proteins, dyes, fibers, dust,et al), which would be enriched and inhibit the following operation of PCR.15 touch DNA samples extracted by chelex-100 were concentrated with Microcon 100, the effect of DNA recovery was different. The same initial volume of 100μl was concentrated to the volume of 8-25μl with an average of 15μl. After dealing with column, DNA concentration was increased by approximately 5 folds, and the average recovery rate of DNA is 61%. But the IPC Ct values were not decreased significantly. And the cost of Microcon column method is also higher than that of magnetic beads method. Thus, the applications of Microcon 100 for touch DNA extraction and purification is limited. It is only suitable for purification and concentration of touch DNA with little pollution.
The results could help to standardize the touch DNA collecting and detecting and optimize the extraction and purification method of touch DNA according to samples and laboratory equipments. It could help to advance success rate of touch DNA test and improve the value of touch DNA as forensic evidence.
如何从微量接触检材中提取到足够的DNA以满足法医学检验的需要,选择合适的提取方法非常关键,是法医物证检验工作者最关注的难题之一。接触DNA检材与常见的生物检材如血痕、精斑相比,属于疑难生物检材。首先,肉眼观察很难判断接触细胞在载体上的具体部位,盲目提取难以获得所需靶细胞；其次,擦拭面积太大会降低靶DNA的浓度,并带来外源物质的污染和干扰；第三,接触DNA检材含有的DNA往往是微量的,属于低拷贝数目(Low copy number,LCN)模板,即“DNA含量少于l00pg,相当于15个双倍体或30个单倍体细胞的生物样本”。因此接触DNA检验不同于常规生物检材的DNA检验,更需系统研究。
目前关于接触DNA提取纯化方法的文献很多,概括起来有Chelex法、磁珠法、有机溶剂法(有机法)、过柱法、硅珠法、硅胶膜法等。但多为针对某种方法对某种类型接触DNA的提取或案例报道,对不同提取纯化方法获得的现场不同类型的接触DNA的质与量的横向比较缺乏系统研究。本研究采用Quantifiler人类DNA定量试剂盒(AB, USA)实时定量技术对目前常用的提取纯化方法包括Chelex法、磁珠提取法(DNA IQ磁珠法、EQ国产磁珠法)、有机溶剂提取法、Microcon过柱法提取的接触DNA进行定量,同时用Identifiler或Sinofiler复合扩增系统(AB,USA)扩增,在AB 3130遗传分析仪上进行STR (short tandem repeat,短串联重复)分型。综合DNA定量和STR分型结果,对各种提取纯化方法获得的DNA的质和量进行评估和系统比较,以建立有效的接触DNA提取纯化方法。
1. Chelex法提取接触DNA的实时定量研究
对使用后放置1个月以内的30个饮料瓶口或吸管、30个矿泉水瓶(杯)口分别采用不洗涤直接加Chelex-100提取与洗涤+Chelex-100的方法提取DNA,通过PCR定量和STR检测,比较2种处理方式获得的DNA平均含量、IPC Ct值、STR检验成功率；对使用后放置1个月以内的10个新鲜饮料瓶口或吸管,使用后分别放置1个月和放置2个月的20把牙刷、20枚烟蒂,分别采用洗涤+Chelex-100和洗涤+Chelex-100+蛋白酶K(PK)的方法提取DNA,通过PCR定量和STR检测,比较2种处理方式获得的DNA平均含量、IPC Ct值、STR检验成功率。研究结果表明,Chelex-100提取前加浸泡洗涤的步骤,可去除部分可溶性杂质,有利于提高获得的DNA量和STR检验成功率,同时降低反映PCR抑制物存在的IPC Ct值。室温放置1个月左右的新鲜接触DNA检材用Chelex法提取时,是否加蛋白酶K对提取的DNA量和STR检验成功率无显著影响；室温放置2个月以上的接触DNA检材用Chelex法提取时,加蛋白酶K消化后获得的DNA量和STR检验成功率均高于不加蛋白酶K的样品,差异有显著性意义。本研究对180份10种实际案件的接触DNA检材用Chelex法提取,烟蒂获得的平均DNA含量最高达175.85 ng,剃须刀较低为7.80 ng。除烟蒂、口香糖采用常规体系提取外,其它检材采用小体系提取,平均DNA浓度均在0.2ng/μl以上,采用2μll模板复合扩增,除衣服、剃须刀的STR分型成功率较低为50%外,其它检材的STR分型成功率均在60%以上。
2.磁珠法提取接触DNA的实时定量研究
对烟蒂、牙刷、手套等3种接触DNA检材各10个分别采用95℃裂解液直接裂解、70℃裂解液直接裂解和预消化(TNE+SDS+PK)等3种前处理方式后用DNA IQ磁珠(Promega, USA)提取DNA,测定并比较三种前处理方式后用DNAIQ磁珠法提取获得的DNA平均含量和IPCCt值。研究结果表明,接触DNA检材采用先加TNE、SDS、PK等消化,然后再用磁珠纯化的方法获得的DNA量高于单纯用裂解液裂解的方法提取的DNA量,有助于提高微量和污染接触DNA检材的STR检验成功率。但该处理方法操作相对繁琐,耗时长,除了污染严重的微量接触DNA检材外,大部分接触DNA检材单纯采用直接裂解法就可以获得足以进行STR分析的DNA。裂解温度方面,95℃裂解与70℃裂解的DNA获得量无显著性差异。对151份8种实际案件的接触DNA检材采用95℃直接裂解的DNAIQ磁珠法在MaxwellTM 16自动仪上提取DNA。结果表明除果核平均DNA获得量为9.51ng以外,其它7种接触检材的平均DNA获得量均大于10ng。采用30μl的小洗脱体积,平均DNA浓度可达0.3ng/μl,采用8μl体系3μl模板扩增,检验成功率最低的果核也达60%。EQ国产磁珠法与DNA IQ磁珠法相比,不仅价格便宜,还少了1个裂解液洗涤和1个洗涤液洗涤的步骤,操作更为简单,但对接触DNA的提取效率无显著性差异。对138份6种实际案件的接触DNA检材在自动化工作站经EQ国产磁珠法提取DNA,采用8μl体系3μl模板扩增,除对饮料瓶口的检验成功率较低,为60%外,对烟蒂、口香糖、手套等接触DNA检材的检验成功率也能达到70%以上。因此也适合接触DNA的提取。
3.5种接触DNA检材提取纯化方法的比较
将稀释为lOng、100ng的标准品DNA,分别采用Chelex法、DNA IQ磁珠法、EQ国产磁珠法、有机法、Microcon过柱法等5种DNA提取纯化方法处理,然后进行PCR定量,比较5种DNA提取纯化方法对标准品DNA的回收率。对烟蒂和牙刷2种常见的接触DNA检材分别采用Chelex法、95℃直接裂解的DNA IQ磁珠法和EQ国产磁珠法提取DNA,通过PCR定量和STR检测分析,比较3种提取方法提取的DNA平均含量、IPC Ct值和STR检验效果；对30例污染严重的接触DNA检材,先用TNE、SDS、PK消化,然后将消化液平均分成2份,1份用DNA IQ磁珠法纯化,1份用有机法抽提,通过PCR定量和STR检测分析,比较磁珠法与有机法对接触DNA的纯化效果；将Chelex法提取的15例5种接触DNA溶液用Microcon100超滤柱进行纯化浓缩,通过PCR定量和STR检测分析,比较Microcon过柱法浓缩前后的浓度、体积、IP Ct值及回收率。结果表明,Chelex法对DNA的提取无损失,磁珠法、有机法、Microcon过柱法对DNA的提取纯化均有不同程度的损失。5种提取纯化方法对DNA的回收率从高到低大致为Chelex法>Microcon过柱法>DNA IQ磁珠法>EQ国产磁珠法>有机法。Chelex法提取DNA,操作简单快速,整个提取过程均在一个离心管中进行,避免了DNA在提取过程中的损耗,对污染轻、杂质少的接触DNA检材,用Chelex法提取最为方便快捷。但Chelex法不能去除DNA溶液中的杂质和降解的DNA碎片,因此,Chelex法提取的陈旧接触检材的DNA进行STR扩增时,即使模板量在试剂盒推荐的范围内,也容易出现等位基因扩增不平衡的现象。磁珠法与Chelex法相比,虽然提取过程中会造成DNA的损失,但提取的DNA纯度高,能有效去除100bp以下的DNA小片段和样品中的色素、蛋白质等杂质,因此,STR扩增时峰高比较均衡,等位基因扩增不平衡的现象不明显。磁珠法与有机法对接触DNA的回收量和IPC Ct值虽然无显著性差异,但磁珠法避免了有机溶剂对操作者的潜在危害,操作简单,耗时短,因此,更适合微量、污染检材的DNA提取及自动化操作。Microcon过柱法操作简单,但对接触DNA的回收效果不一,100μl的起始体积,过柱浓缩后的体积在8-25μl之间,平均为15μl。过柱后,DNA浓度平均约增加5倍,对DNA的回收率平均为61%,但反映PCR抑制物存在的IPC Ct值无显著降低。同时,Microcon过柱法的成本也高于磁珠法。因此,Microcon过柱法对接触DNA的提取纯化应用有限,只适合污染轻的接触DNA的纯化浓缩。
本研究通过对5种常见的提取纯化方法获得的DNA的质和量进行系统比较和评估,为规范法医检验中接触DNA检材的提取送检以及法医工作者如何根据检材、实验室设备等情况选择合适有效的接触DNA提取纯化方法提供了科学依据,有利于提高接触DNA检材的检验成功率,充分发挥接触DNA检材的证据价值。
Touch DNA analysis is one of the difficulties and hotspots of forensic DNA typing. The touch DNA samples in forensic DNA typing means the objects with sloughed off skin and mucous membrane cells. The objects which contacted with human skin or mucous membrane are likely to contain exfoliated epithelial cells. The biological evidence not only exists on the crime scene but also can be found on the clothing, utensils and other objects of criminal suspects or the victims. As criminals' anti-surveillance means become increasingly strong, obvious biological evidence (such as sperm spots, blood stains, hair, human tissue, etc.) are often purposely destroyed. Small amount epithelial cells from the body are often neglected, such as the oral exfoliated epithelial cells left on cigarette butts, beverage cans, fruit-stones, chewing gums, the skin shedding epithelial cells left in the gloves, clothes, hats, fingerprints and so on. However, it could be a vital clues to determine the nature of the case and provide scientific evidence for the court, once the touch DNA from these potential minute biological evidence can be typed successfully.
To satisfy the needs of the forensic case detection, it's very important to select the appropriate DNA extraction and purification method for obtaining sufficient DNA from the touch cells which are minute. Touch DNA samples belong to difficult samples in comparison of to common biological samples such as bloodstains, semen stains. The reasons are the following:First, it is difficult to judge the exact location on the carrier and obtain target cells only by naked eyes; Second, it will decrease target DNA concentration and bring exogenous pollution and disturbance when extracted with more wiped area; Third, touch DNA samples often contain low copy number (LCN) templates, which means "DNA amount is less than 100pg, equivalent to 15 diploid or 30 haploid cell. " Therefore, touch DNA testing is different from the conventional biological samples and needs to study systemically.
Nowadays, there are numerous references on touch DNA extraction and purification methods. In general, there are the Chelex-100 method, the magnetic beads method, the organic method (phenol-chloroform method), spin column method, the silica beads method, the silica membrane method, and so on. But mostly references only aimed at certain method of DNA extraction about certain types of touch DNA or simply case reports. There are fewer horizontal comparisons of DNA quality and quantity among the different extraction and purification methods. In this study, DNA extracted and purified by the Chelex-100 method, the magnetic beads method (DNA IQ system and EQ system), organic extraction and the Microcon100 column (Millipore, USA) method was quantified by the real-time PCR with Quantifiler Human DNA Quantification kit. STR typing with IdentifierTM or SinofilerTM multiplex STR amplification system and AB 3130 genetic analyzer(AB, USA) were also used to detect the quality of extracted touch DNA. By systematic comparison between quantification and STR typing, proper experimental evidence could help the r standardization of collecting touch DNA evidence and determination on how to select an appropriate and effective method of extraction and purification.
The results are as follows:
1. Real time quantificational study of DNA extracted by Chelex-100 method from touch DNA samples
DNA was extracted from the familiar touch DNA samples(beverage cans or sucker, cigarette butts, toothbrushes) respectivel by no-washing Chelex-100 method, washing+Chelex-100 method and washing+Chelex-100+proteinase K method. The DNA average amounts, IPC Ct and success rate of STR typing were observed and compared by PCR quantification and STR analysis. The results are as follows: The steps of immersion washing in Chelex-100 extraction can remove some of the soluble inhibitor, which can help increasing DNA quality, improving STR typing and reducing the IPC Ct which could reflect the PCR inhibitor of existing. For fresh epithelial cell samples keeped at room temperature in about 1 month, there was no significant impact on DNA yield and STR typing by Chelex method, whether plus proteinase K or not. But for more than 2 months of epithelial cell samples, it would increase more DNA yield and STR typing with proteinase K addition in Chelex-100 extraction than without. The difference was significant. In 180 cases of touch DNA extracted by Chelex method, the average DNA yield from cigarette butts was highest (175.85ng) and the average DNA yield from shavers was lower(7.80ng). Except cigarette butts, chewing gums and toothbrushs were extracted by standard volume chelex-100 protocol, other samples were for little volume chelex-100 method. The average DNA concentration for 180 samples was beyond 0.2ng/μl. The success rates of STR typing for clothes and shavers were 50%, and the success rates of STR typing for other touch DNA samples were over 60% with 2μl templet.
2. Real time quantificational study of DNA extracted by the magnetic beads method from touch DNA samples
DNA was extracted from the 3 touch DNA samples(10 cigarette butts,10 toothbrushes and 10 gloves) by DNA IQ system respectively after three preprocess protocols(95℃direct lysis,70℃direct lysis and digesting with TNE, SDS, and proteinase K). The DNA average amounts, IPC Ct and success rate of STR typing were observed and compared between PCR quantification and STR analysis. The results are as follows:It yields more DNA by digesting samples with TNE, SDS, and proteinase K before the magnetic beads purification than using direct lysis protocol, which could increase STR typing for minute and polluted smples. However, this preprocess protocol is relatively cumbersome and time-consuming. Except seriously polluted and minute DNA samples, most touch DNA samples could get enough DNA for STR analysis only by direct lysis protocol. As to lysis temperature, there is no significant difference between 95℃and 70℃in the quantity of DNA yield. After using direct lysis (95℃) protocol,151 cases of the eight kinds of touch DNA samples were extracted DNA in MaxwellTM 16 by DNA IQ magnetic beads methods (Promega). DNA quantification result shew that the average DNA yields were more than lOng for each touch DNA, except the DNA from fruit-stones,whose average yield was 9.51ng. Using 30μl elution volume, the average DNA concentrations could come up to 0.3ng/μl, and the success rate of STR typing from fruit-stones could reach to 60% with 3μl templet in 8μl amplification system. Compared with the DNA IQ System, the EQ magnetic beads kit made in China is not only more simple and much cheaper, but also lack of a lysis buffer washing and a wash buffer washing steps. But there is no significant difference in DNA extraction efficiency.138 cases touch DNA samples were extracted DNA in automation workstation by the EQ magnetic beads methods. Except the lower success rate of STR typing from beverage cans (60%), the success rate from cigarette butts, chewing gums, and gloves were about 70% with 3μl templet in 8μl amplification system. So the EQ magnetic beads kit is also suitable for touch DNA extraction.
3. The comparison of 5 touch DNA extraction and purification methods
The standard DNA diluted to 10ng, 100ng was treated respectively by 5 touch DNA extraction and purification methods(the Chelex-100 method, DNA IQ system, the EQ magnetic beads method, organic extraction and the Microcon100 column method). The recovery rates of standard DNA treated with 5 methods were compared by PCR quantification. DNA samples were extracted from cigarette butts and toothbrushes the 2 touch DNA samples respectively by Chelex method,95℃direct lysis DNA IQ method and the EQ magnetic beads methods. The DNA average amounts, IPC Ct and success rate of STR typing were compared by PCR quantification and STR analysis. The DNA samples were purified respectively by DNA IQ system and the organic method from the 30 polluted touch DNA samples after digesting by TNE, SDS and PK. The purification effects were compared by PCR quantification and STR analysis. The 15 touch DNA samples treated by the Chelex method were purified and concentrated by Microcon 100. The purification and concentration effects were compared by PCR quantification and STR analysis. The results are as follows:In general, there isn't any loss of DNA with the Chelex method to extract it. However, other protocols of DNA extraction and purification, such as the magnetic beads method, the organic method and the Microcon 100 column method, would lose some DNA. Comparatively, the rate of DNA recovery by those methods can be ranked as:Chelex> Microcon> DNA IQ System> EQ magnetic beads method (made in China)>Organic extraction. The Chelex method is simple and fast, the process of which is carried out in a centrifuge tube so that the loss of the DNA in extraction process was avoided. So the Chelex method is preferred to extract touch DNA samples with less pollution and low impurity. The magnetic beads method could result in the loss of the DNA, but yield DNA of high purity. It could concentrate DNA by adjusting elution volume and it is suitable for automatic operations. Organic extraction method is the classic method of DNA extraction. Organic method may obtain 200bp-1000kb genome DNA, the purity of which is high. DNA was extracted from 30 cases of polluted touch DNA samples by DNA IQ System and the organic method, there was no significant difference of the DNA recovery rate and IPC Ct values of DNA extracred by DNA IQ System and the organic method. But the magnetic beads method could avoid the potential hazard of operator caused by organic solvents, more simple operation and timesaving. In conclusion, the magnetic beads method is more suitable for minute and polluted samples of DNA extraction and automation. So the magnetic beads method become the first choice for minute and polluted DNA samples. The operations of Microcon100 column method is simple, but it could not filtered macromolecular impurities(such as proteins, dyes, fibers, dust,et al), which would be enriched and inhibit the following operation of PCR.15 touch DNA samples extracted by chelex-100 were concentrated with Microcon 100, the effect of DNA recovery was different. The same initial volume of 100μl was concentrated to the volume of 8-25μl with an average of 15μl. After dealing with column, DNA concentration was increased by approximately 5 folds, and the average recovery rate of DNA is 61%. But the IPC Ct values were not decreased significantly. And the cost of Microcon column method is also higher than that of magnetic beads method. Thus, the applications of Microcon 100 for touch DNA extraction and purification is limited. It is only suitable for purification and concentration of touch DNA with little pollution.
The results could help to standardize the touch DNA collecting and detecting and optimize the extraction and purification method of touch DNA according to samples and laboratory equipments. It could help to advance success rate of touch DNA test and improve the value of touch DNA as forensic evidence.
引文
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