接触DNA检材提取纯化方法的比较及法医学应用
发布时间：2018-08-11 21:09
【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。
[Abstract]:Contact DNA testing is one of the difficulties and hotspots in forensic DNA testing. Contact DNA testing in forensic identification refers to the testing materials containing exfoliated cells of human skin and mucosa, including those containing oral exfoliated cells, exfoliated cells on the surface of the body, human excreta or secretions, etc. All objects may leave the contact's exfoliated cells. These exfoliated evidences can be found at the scene of a crime, as well as on objects such as clothing and utensils of the suspect and the victim. Destruction and destruction, whereas metabolic exfoliated cells from the human body are often neglected due to small amounts, such as exfoliated oral epithelial cells left on cigarette butts, beverage cans, fruit pits, chewing gum, gloves, clothing, hats, fingerprints, skin exfoliated epithelial cells, and so on. DNA typing of key material evidence can often become a key clue to the detection of a case and provide scientific evidence for the qualitative analysis of a case and the sentencing of a court.
How to extract enough DNA from micro-contact samples to meet the needs of forensic medical examination is very important to select the appropriate extraction method, which is one of the most concerned problems for forensic medical evidence inspectors. It is difficult to obtain the desired target cells blindly when contacting the specific parts of the cell on the carrier; secondly, too large wiping area will reduce the concentration of target DNA, and bring about contamination and interference of foreign substances; thirdly, the DNA contained in the DNA samples is often micro, belonging to the low copy number (LCN) template, that is, "DNA content is less than l0." 0 pg, equivalent to 15 diploid or 30 haploid cells in biological samples.
At present, there are many literatures about the methods of extracting and purifying contact DNA, such as Chelex method, magnetic beads method, organic solvent method (organic method), column method, silica beads method, silica gel membrane method, etc. In this study, Quantifiler Human DNA Quantitative Kit (AB, USA) was used to quantify the contact DNA extracted by Chelex method, magnetic beads extraction method (DNA IQ beads, EQ beads method), organic solvent extraction method and MicroCon column method. STR (short tandem repeat) typing was performed on AB 3130 genetic analyzer by using Identifier or Sinofiler amplification system (AB, USA). The quality and quantity of DNA obtained by various extraction and purification methods were evaluated and compared systematically by combining the results of DNA quantification and STR typing.
Real time quantitative study on Extraction of contact DNA by 1. Chelex method
DNA was extracted from 30 beverage bottles (cups) and 30 mineral water bottles (cups) which were placed within one month after use by adding Chelex-100 extraction without washing and washing + Chelex-100 extraction respectively. The average DNA content, IPCCT value and success rate of STR test were compared by PCR quantitative analysis and STR detection. Ten fresh beverage bottles or straws within one month were placed for one month and 20 toothbrushes and 20 cigarette butts for two months. DNA was extracted by washing + Chelex-100 and washing + Chelex-100 + protease K (PK) respectively. The average DNA content, IPCCT value and STR test were compared by PCR and STR. The results showed that the procedure of soaking and washing before extraction of Chelex-100 could remove some soluble impurities, improve the amount of DNA obtained and the success rate of STR test, and reduce the IPCCT value reflecting the presence of PCR inhibitors. There was no significant difference in DNA content and the success rate of STR test. When exposed DNA samples were extracted by Chelex method at room temperature for more than 2 months, the amount of DNA digested by protease K and the success rate of STR test were higher than those without protease K. The results showed that the difference was significant. The average DNA content of tobacco stem was 175.85 ng, and that of shaver was 7.80 ng. Except for tobacco stem and chewing gum, the other samples were extracted by small system. The average DNA concentration was above 0.2 ng/mul. The two-mull template was used to amplify the DNA. The success rate of SRT typing of clothing and shaver was 50% lower than that of other samples. The success rate of TR typing was over 60%.
Real time quantitative study of 2. magnetic beads extraction for contact DNA
DNA was extracted by DNA IQ magnetic beads (Promega, USA) after three pretreatments, namely, direct cleavage at 95 C, direct cleavage at 70 C and pre-digestion (TNE + SDS + PK) at 70 C. The average DNA content obtained by DNA IQ magnetic beads and IPCC after three pretreatments were determined and compared. The results showed that the amount of DNA obtained by adding TNE, SDS, PK and other digestion methods and then purifying by magnetic beads was higher than that by using lysate lysate alone, which was helpful to improve the success rate of STR test for micro and contaminated DNA samples. In addition to the severely contaminated DNA samples, most of them could obtain enough DNA for STR analysis by direct cleavage. At the cleavage temperature, there was no significant difference in the amount of DNA obtained by cleavage at 95 C and 70 C. DNA was extracted by ellTM 16 automaton. The results showed that the average DNA yield of the other seven contact samples was more than 10 ng except the average DNA yield of 9.51 ng. The average DNA concentration could reach 0.3 ng/mul with a small elution volume of 30 ml, and the lowest success rate was 60% with a 3-mul template amplification in 8-mul system. Compared with the method of IQ magnetic beads, it is not only cheaper, but also less than one washing step of lysate and one washing liquid. The operation is simpler, but there is no significant difference in the efficiency of DNA extraction. The detection success rate of beverage bottle mouth is lower than 60%. The detection success rate of tobacco butt, chewing gum, gloves and other DNA samples can reach more than 70%.
Comparison of 3.5 methods for extraction and purification of contact DNA samples
DNA samples diluted to lOng and 100ng were extracted and purified by Chelex method, DNA IQ magnetic beads method, EQ domestic magnetic beads method, organic method and Microcon column-passing method, and then quantified by PCR. The recovery rates of DNA samples were compared between the five DNA extraction and purification methods. DNA was extracted by ex method, DNA IQ magnetic bead method and EQ domestic magnetic bead method. The average DNA content, IPC CT value and STR test results of the three methods were compared by PCR quantitative analysis and STR analysis. For 30 seriously contaminated DNA samples, TNE, SDS and PK were used to digest, and then the digestive juice was divided into two parts, one part was used DNA IQ magnetic bead. Methods: One sample was extracted by organic method, and the purifying effect of magnetic bead method and organic method was compared by PCR quantitative analysis and STR detection analysis. Five kinds of contact DNA solution extracted by Chelex method were purified and concentrated by Microcon 100 ultrafiltration column, and the concentration, volume, IP of MicroCon before and after concentration were compared by PCR quantitative analysis and STR detection analysis. The results showed that there was no loss in the extraction of DNA by Chelex method, the loss of DNA extraction and purification by magnetic beads, organic method and MicroCon column-passing method was different. The recovery of DNA by five extraction and purification methods from high to low was approximately the same as that by Chelex method, MicroCon column-passing method, DNA IQ magnetic beads method and EQ domestic magnetic beads method. The whole extraction process is carried out in a centrifugal tube, which avoids the loss of DNA in the extraction process. Chelex method is the most convenient and fast method for the detection of light contamination and less impurities. However, Chelex method can not remove impurities in DNA solution and degraded DNA fragments. The unbalanced amplification of alleles is easy to occur even when the template size is within the recommended range of the kit. Compared with the Shell method, the magnetic bead method may cause the loss of DNA, but the purity of the extracted DNA is high, which can effectively remove the small fragments of DNA below 100 bp and the pigment, protein and other impurities in the sample. Although there was no significant difference between magnetic bead method and organic method in the recovery of DNA and IPCCT value, magnetic bead method avoided the potential harm of organic solvents to operators. It was simple and time-consuming. Therefore, magnetic bead method was more suitable for DNA extraction of trace and contaminated samples. Microcon over-column method is easy to operate, but its recovery effect is different. The initial volume of 100 ml is between 8-25 ml, and the average volume of 15 ml. After passing through the column, the average DNA concentration increases about 5 times, and the average recovery rate of DNA is 61%. However, the IPCCT value reflecting the presence of PCR inhibitors has not decreased significantly. The cost of the MicroCon method is also higher than that of the magnetic bead method. Therefore, the application of the MicroCon method to the extraction and purification of contact DNA is limited, and it is only suitable for the purification and concentration of light contaminated contact DNA.
This study systematically compares and evaluates the quality and quantity of DNA obtained by five common methods of extraction and purification, which provides scientific basis for standardizing the extraction and inspection of contact DNA samples in forensic examination and how forensic workers choose appropriate and effective methods of extraction and purification of contact DNA according to the materials and laboratory equipment. We should improve the success rate of inspection of DNA specimens and give full play to the evidence value of contacting DNA specimens.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919.4
本文编号：2178241
[Abstract]:Contact DNA testing is one of the difficulties and hotspots in forensic DNA testing. Contact DNA testing in forensic identification refers to the testing materials containing exfoliated cells of human skin and mucosa, including those containing oral exfoliated cells, exfoliated cells on the surface of the body, human excreta or secretions, etc. All objects may leave the contact's exfoliated cells. These exfoliated evidences can be found at the scene of a crime, as well as on objects such as clothing and utensils of the suspect and the victim. Destruction and destruction, whereas metabolic exfoliated cells from the human body are often neglected due to small amounts, such as exfoliated oral epithelial cells left on cigarette butts, beverage cans, fruit pits, chewing gum, gloves, clothing, hats, fingerprints, skin exfoliated epithelial cells, and so on. DNA typing of key material evidence can often become a key clue to the detection of a case and provide scientific evidence for the qualitative analysis of a case and the sentencing of a court.
How to extract enough DNA from micro-contact samples to meet the needs of forensic medical examination is very important to select the appropriate extraction method, which is one of the most concerned problems for forensic medical evidence inspectors. It is difficult to obtain the desired target cells blindly when contacting the specific parts of the cell on the carrier; secondly, too large wiping area will reduce the concentration of target DNA, and bring about contamination and interference of foreign substances; thirdly, the DNA contained in the DNA samples is often micro, belonging to the low copy number (LCN) template, that is, "DNA content is less than l0." 0 pg, equivalent to 15 diploid or 30 haploid cells in biological samples.
At present, there are many literatures about the methods of extracting and purifying contact DNA, such as Chelex method, magnetic beads method, organic solvent method (organic method), column method, silica beads method, silica gel membrane method, etc. In this study, Quantifiler Human DNA Quantitative Kit (AB, USA) was used to quantify the contact DNA extracted by Chelex method, magnetic beads extraction method (DNA IQ beads, EQ beads method), organic solvent extraction method and MicroCon column method. STR (short tandem repeat) typing was performed on AB 3130 genetic analyzer by using Identifier or Sinofiler amplification system (AB, USA). The quality and quantity of DNA obtained by various extraction and purification methods were evaluated and compared systematically by combining the results of DNA quantification and STR typing.
Real time quantitative study on Extraction of contact DNA by 1. Chelex method
DNA was extracted from 30 beverage bottles (cups) and 30 mineral water bottles (cups) which were placed within one month after use by adding Chelex-100 extraction without washing and washing + Chelex-100 extraction respectively. The average DNA content, IPCCT value and success rate of STR test were compared by PCR quantitative analysis and STR detection. Ten fresh beverage bottles or straws within one month were placed for one month and 20 toothbrushes and 20 cigarette butts for two months. DNA was extracted by washing + Chelex-100 and washing + Chelex-100 + protease K (PK) respectively. The average DNA content, IPCCT value and STR test were compared by PCR and STR. The results showed that the procedure of soaking and washing before extraction of Chelex-100 could remove some soluble impurities, improve the amount of DNA obtained and the success rate of STR test, and reduce the IPCCT value reflecting the presence of PCR inhibitors. There was no significant difference in DNA content and the success rate of STR test. When exposed DNA samples were extracted by Chelex method at room temperature for more than 2 months, the amount of DNA digested by protease K and the success rate of STR test were higher than those without protease K. The results showed that the difference was significant. The average DNA content of tobacco stem was 175.85 ng, and that of shaver was 7.80 ng. Except for tobacco stem and chewing gum, the other samples were extracted by small system. The average DNA concentration was above 0.2 ng/mul. The two-mull template was used to amplify the DNA. The success rate of SRT typing of clothing and shaver was 50% lower than that of other samples. The success rate of TR typing was over 60%.
Real time quantitative study of 2. magnetic beads extraction for contact DNA
DNA was extracted by DNA IQ magnetic beads (Promega, USA) after three pretreatments, namely, direct cleavage at 95 C, direct cleavage at 70 C and pre-digestion (TNE + SDS + PK) at 70 C. The average DNA content obtained by DNA IQ magnetic beads and IPCC after three pretreatments were determined and compared. The results showed that the amount of DNA obtained by adding TNE, SDS, PK and other digestion methods and then purifying by magnetic beads was higher than that by using lysate lysate alone, which was helpful to improve the success rate of STR test for micro and contaminated DNA samples. In addition to the severely contaminated DNA samples, most of them could obtain enough DNA for STR analysis by direct cleavage. At the cleavage temperature, there was no significant difference in the amount of DNA obtained by cleavage at 95 C and 70 C. DNA was extracted by ellTM 16 automaton. The results showed that the average DNA yield of the other seven contact samples was more than 10 ng except the average DNA yield of 9.51 ng. The average DNA concentration could reach 0.3 ng/mul with a small elution volume of 30 ml, and the lowest success rate was 60% with a 3-mul template amplification in 8-mul system. Compared with the method of IQ magnetic beads, it is not only cheaper, but also less than one washing step of lysate and one washing liquid. The operation is simpler, but there is no significant difference in the efficiency of DNA extraction. The detection success rate of beverage bottle mouth is lower than 60%. The detection success rate of tobacco butt, chewing gum, gloves and other DNA samples can reach more than 70%.
Comparison of 3.5 methods for extraction and purification of contact DNA samples
DNA samples diluted to lOng and 100ng were extracted and purified by Chelex method, DNA IQ magnetic beads method, EQ domestic magnetic beads method, organic method and Microcon column-passing method, and then quantified by PCR. The recovery rates of DNA samples were compared between the five DNA extraction and purification methods. DNA was extracted by ex method, DNA IQ magnetic bead method and EQ domestic magnetic bead method. The average DNA content, IPC CT value and STR test results of the three methods were compared by PCR quantitative analysis and STR analysis. For 30 seriously contaminated DNA samples, TNE, SDS and PK were used to digest, and then the digestive juice was divided into two parts, one part was used DNA IQ magnetic bead. Methods: One sample was extracted by organic method, and the purifying effect of magnetic bead method and organic method was compared by PCR quantitative analysis and STR detection analysis. Five kinds of contact DNA solution extracted by Chelex method were purified and concentrated by Microcon 100 ultrafiltration column, and the concentration, volume, IP of MicroCon before and after concentration were compared by PCR quantitative analysis and STR detection analysis. The results showed that there was no loss in the extraction of DNA by Chelex method, the loss of DNA extraction and purification by magnetic beads, organic method and MicroCon column-passing method was different. The recovery of DNA by five extraction and purification methods from high to low was approximately the same as that by Chelex method, MicroCon column-passing method, DNA IQ magnetic beads method and EQ domestic magnetic beads method. The whole extraction process is carried out in a centrifugal tube, which avoids the loss of DNA in the extraction process. Chelex method is the most convenient and fast method for the detection of light contamination and less impurities. However, Chelex method can not remove impurities in DNA solution and degraded DNA fragments. The unbalanced amplification of alleles is easy to occur even when the template size is within the recommended range of the kit. Compared with the Shell method, the magnetic bead method may cause the loss of DNA, but the purity of the extracted DNA is high, which can effectively remove the small fragments of DNA below 100 bp and the pigment, protein and other impurities in the sample. Although there was no significant difference between magnetic bead method and organic method in the recovery of DNA and IPCCT value, magnetic bead method avoided the potential harm of organic solvents to operators. It was simple and time-consuming. Therefore, magnetic bead method was more suitable for DNA extraction of trace and contaminated samples. Microcon over-column method is easy to operate, but its recovery effect is different. The initial volume of 100 ml is between 8-25 ml, and the average volume of 15 ml. After passing through the column, the average DNA concentration increases about 5 times, and the average recovery rate of DNA is 61%. However, the IPCCT value reflecting the presence of PCR inhibitors has not decreased significantly. The cost of the MicroCon method is also higher than that of the magnetic bead method. Therefore, the application of the MicroCon method to the extraction and purification of contact DNA is limited, and it is only suitable for the purification and concentration of light contaminated contact DNA.
This study systematically compares and evaluates the quality and quantity of DNA obtained by five common methods of extraction and purification, which provides scientific basis for standardizing the extraction and inspection of contact DNA samples in forensic examination and how forensic workers choose appropriate and effective methods of extraction and purification of contact DNA according to the materials and laboratory equipment. We should improve the success rate of inspection of DNA specimens and give full play to the evidence value of contacting DNA specimens.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919.4
【引证文献】
相关期刊论文 前2条
1 曲春冰;张国翔;袁家龙;;KingFisher磁珠纯化仪在提取脱落细胞检材DNA上的应用[J];广东公安科技;2011年01期
2 焦伟;刘斐;谭毅;;人短串联重复序列相关技术及其在法医学中的应用研究进展[J];中国临床新医学;2011年11期
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