全基因组扩增方法的建立及其在法医学中的应用

发布时间：2018-04-16 07:29

本文选题：全基因组扩增 + 多重置换扩增反应　；参考：《兰州大学》2008年硕士论文

【摘要】： 研究背景与目的在法医学检案实践中,常遇到样本DNA含量极其有限,以致DNA分型无法完成或不能满足重复检测的需要。因此,对微量模板DNA分析便成为法医学DNA检验的一个难题。以PCR-STR为代表的遗传标记分析技术虽然一定程度上解决了法医学微量检材的难题,但一些检材仍因DNA含量极其有限而无法检测成功,或仅检出少数基因座而无法使累计鉴别能力达到认定水平。这类检材被称为痕量DNA,也称低拷贝模板(low copy number,LCN),Gill等将其定义为低于100pg的模板DNA。为解决这一难题,有研究用增加PCR循环数,但模板DNA量太低时易产生等位基因丢失或非特异带,且循环数增加到一定程度后再增加循环数也不会显著增加灵敏度;另外,有人试图用巢式PCR,但巢式PCR技术仅限于单一位点分析,无法提高目前法医学中常规DNA检测所用的多位点复合扩增所需的模板数量。全基因组扩增(Whole Genome Amplification,WGA)是一种能从有限的DNA样品获得充足的DNA量供后续分析的技术,其基本思路是通过对微量组织甚至单个细胞的整个基因组DNA扩增,大幅度增加DNA的总量。因此被认为是目前可以解决上述难题的一种有效方法。 WGA技术经10余年探索,在方法上已逐步发展和不断完善起来,已被广泛用于疾病基因研究等领域,并取得了良好的效果。但在法医学方面的应用很少。在目前常用的几种WGA方法中,改良扩增前引物延伸方法(Improved primerextension preamplification,IPEP)和多重置换扩增方法(Multiple displacementamplification,MDA)对STR基因座分型显示出较好的效果。故本研究探讨了这两种方法用于法医学微量检材的效果,包括对其灵敏度、扩增忠实性、能否用于法医学常用STR基因座及常用检材等进行系统性研究,从而对其用于法医学实践的可行性进行评价,并建立稳定可行的技术方案。方法 1.对30例口腔拭子样品,采用酚-氯仿法提取DNA,实时荧光定量PCR技术定量,并分别稀释成1ng、0.5ng、0.1ng、0.05ng、0.025ng、0.01ng六种模板量DNA,然后进行MDA反应和IPEP反应。采用实时荧光定量PCR技术检测MDA方法和IPEP方法的产物浓度,比较两种方法的扩增效率。分别以MDA产物和IPEP产物为模板DNA,用AmpFLSTR~(?)Identifiler~(?)试剂盒扩增,ABI3100基因分型仪检测基因型,比较MDA产物和IPEP产物用于STR分型的效果。 2.对IPEP方法反应体系的成分和终浓度进一步优化,建立改良IPEP方法,并检测其扩增效率和STR分型效果。将改良IPEP方法用于30例血液、30例血纱、30例精液等三类法医学常见生物检材进行检测,观察改良IPEP方法的可重复性和对法医学常见生物检材的适用性。 3.将改良IPEP方法用于20例汗潜指印、20例一次性牙刷、20例自然脱落头发等三种法医学常见疑难微量检材的模拟案例,观察改良IPEP方法对上述检材的STR分型效果;用于现场果核10例、矿泉水瓶10例、烟蒂10例、衣领10例等实际案例,观察改良IPEP方法的实际案件检验能力。结果 1.MDA法产量为5.15ug～19.75ug,可对模板DNA增加5.15×10~3～1.98×10~6倍:IPEP法产量为0.5ng～66ng,可对模板DNA增加25～310倍。MDA法产物浓度高于IPEP法,差异有显著性意义(F=3643.433,P＜0.001)。 2.常规检测法、MDA法、IPEP法的基因座检出数有显著性差异(F=1033.297,P＜0.001)。当DNA模板量为1ng时,常规检测方法、MDA方法、IPEP方法均可获得复合扩增系统所有基因座的准确分型结果;当DNA模板量为0.5ng时,常规检测方法和IPEP方法均获得所有基因座的准确分型结果,MDA方法获得10个以上基因座的准确分型结果;当DNA模板量为0.1ng～0.01ng时,MDA方法基因座检出数高于常规检测方法,IPEP方法基因座检出数高于MDA方法。 3.DNA模板量≥1ng,其MDA产物杂合子基因座的等位基因扩增均衡;DNA模板量≤0.5ng,其MDA产物的STR分型图谱可见等位基因不平衡或丢失现象,且模板DNA量越低,等位基因不平衡或丢失现象越明显。DNA模板量≥0.05ng,其IPEP产物杂合子基因座的等位基因扩增均衡;DNA模板量≤0.025ng,其IPEP产物的STR分型图谱可见等位基因不平衡或丢失。MDA产物和IPEP产物用于STR分型时均未观察到非特异产物峰。 4.采用保真性更好的DNA聚合酶和增加随机引物用量,建立了改良IPEP方法。0.1ng、0.05ng、0.025ng、0.01ng四组模板量DNA经改良IPEP法扩增所得产物浓度均高于IPEP法,差异有显著性(F=289.899,P＜0.001)。改良IPEP法对上述模板DNA扩增获得3ng～84ng产物,可使模板DNA增加160～1220倍。DNA模板量为0.025ng时,改良IPEP法可获得完整准确的分型结果。DNA模板量为0.01ng时,改良IPEP法的平均基因座检出数高于IPEP法,部分基因座仍未检出,以D7S820、D18S51、FGA等较常见。改良IPEP方法的产物用于STR分型时,未见非特异产物峰及等位基因不平衡或丢失。 5.改良IPEP方法对0.025ng血液DNA、血纱DNA、精液DNA检测均获得所有基因座的准确分型结果。 6.改良IPEP方法可显著提高汗潜指印DNA(t=5.423,P＜0.001)、一次性牙刷DNA(t=6.835,P＜0.001)的基因座检出数,但对自然脱落毛发DNA的基因座检出数低于常规检测法(t=3.249,P＜0.001)。改良IPEP方法对实际案件中果核、杯口、烟蒂、衣领等检材的检测成功率和平均基因座检出数均高于常规检测方法,且检出STR基因座数均不少于9个。结论 1.MDA法产量高,但灵敏度低,当模板量低于1ng时,产物序列保真性差,影响后续STR分型的准确性。因此,MDA法对于保留样本有效,但不适用于痕量DNA检测。 2.IPEP法灵敏度高,产物序列保真性好,对0.05ng的基因组DNA可获得良好的STR分型效果,但产量低。 3.DNA聚合酶和随机引物用量对IPEP方法有重要影响。采用保真性更好的DNA聚合酶和增加随机引物至一定终浓度,提高了扩增效率。在此基础上建立的改良IPEP方法的产物序列保真性好,且产量和灵敏度都进一步提高,改善了痕量DNA的STR分型效果。 4.改良IPEP方法对于口腔拭子、血液、血纱、精液等不同生物性检材、不同状态的同种检材均获得准确可靠的结果,表明了改良IPEP方法的稳定性好,适用于法医学常见生物性检材。 5.改良IPEP方法可显著提高微量皮肤脱落细胞、口腔粘膜脱落细胞等痕量检材的检测成功率和平均基因座检出数,可使累计鉴别能力达到认定水平,以满足法医学个体识别要求。但改良IPEP方法对自然脱落毛发这类降解的短片段DNA样品效果不佳。
[Abstract]:Research background and purpose
In forensic practice, often encounter the sample DNA content is extremely limited, so that the DNA type can not be completed or can not meet the need of repeated detection. Therefore, the analysis of trace template DNA it becomes a difficult problem in forensic DNA test. Although the problem analysis technology to a certain extent to solve the forensic trace materials of genetic mark represented by PCR-STR, but some samples are still extremely limited because DNA was not detected successfully, or only a few genes can make a positive cumulative discrimination reached that level. These samples are called trace DNA, also known as low copy number (low copy number, LCN Gill), the defined as less than 100PG DNA. template to solve this problem, a study by increasing the number of PCR cycles, but the amount of template DNA is too low to produce allelic loss or non specific bands, and the number of cycles increased to a certain extent after the increase in cycle number Does not significantly increase the sensitivity; in addition, there are people trying to use nested PCR, but nested PCR is limited to a single locus analysis, to improve the conventional DNA multilocus forensic science detection by multiplex amplification the number of templates required. Whole genome amplification (Whole Genome, Amplification, WGA) is a kind of can obtain a sufficient amount of DNA from the limited samples for subsequent analysis of the DNA technology, the basic idea is based on the micro tissue or even a single cell whole genome amplification of DNA, the total increase of DNA. It is considered an effective way to solve the above problems at present.
The technology of WGA through more than 10 years of exploration, the method has been gradually developed and improved, has been widely used in the field of disease gene research, and achieved good results. But in forensic applications rarely. In several of the traditional WGA method, improved primer extension preamplification (Improved primerextension preamplification. IPEP) and multiple displacement amplification (Multiple displacementamplification, MDA) showed better effect on STR genotype. Therefore, this study discusses the two methods for forensic trace materials effect, including the sensitivity and amplification fidelity, can be used for forensic STR locus and used. Material for a systematic study, which is used to evaluate the feasibility of the practice of forensic medicine, and the establishment of the technical scheme is stable and feasible.
Method
1. of the 30 cases of oral swab samples, DNA extracted by phenol chloroform method, real-time fluorescence quantitative PCR and quantitative, were diluted to 1ng, 0.5ng, 0.1ng, 0.05ng, 0.025ng, 0.01ng six kinds of template was DNA, then MDA and IPEP reaction. The product concentration detection real-time PCR measurement MDA method and IPEP method, the amplification efficiency of two methods are compared. Using MDA products and IPEP products as template DNA, AmpFLSTR~ (?) Identifiler~ (?) kit for ABI3100 gene amplification, gene type detection apparatus, in STR type effect compared with MDA products and IPEP products.
2. methods of IPEP reaction components and the final concentration of further optimization, a modified IPEP method, and detect its amplification efficiency and STR type effect. The improved IPEP method for 30 cases of blood, blood of 30 cases with yarn, 30 semen of three forensic common samples were detected for observation of modified IPEP repeatability of the method and the common forensic biological samples.
3. improved IPEP method for 20 cases of sweat latent fingerprints, 20 cases of disposable toothbrush, 20 cases of natural hair shedding simulation of three kinds of common difficult forensic trace materials, observation of modified IPEP method on the samples of the STR type; 10 cases for site stones, 10 cases of mineral water bottles, cigarette butts in 10 cases the actual case, the collar in 10 cases. The actual test case observing ability of modified IPEP method.
Result
The yield of 1.MDA method is 5.15ug ~ 19.75ug, which can increase 5.15 x 10~3 to 1.98 * 10~6 times for template DNA: the yield of IPEP is 0.5ng ~ 66ng, which can increase 25~310 times to template DNA, and the concentration of.MDA product is higher than that of 66ng method. The difference is significant.
2. conventional detection method, MDA method, IPEP method for detection of the number of loci with significant difference (F=1033.297, P < 0.001). When the template DNA was 1ng, the conventional detection method, MDA method, IPEP method can obtain all loci multiplex system for accurate genotyping results; when the template DNA was 0.5ng when the conventional detection method and IPEP method were all loci accurate genotyping results, the MDA method to obtain accurate genotyping results of 10 or more loci; when the template DNA was 0.1ng ~ 0.01ng, MDA loci detected number is higher than that of the conventional detection methods, IPEP method was higher than the number of loci in MDA method.
The template 3.DNA is larger than 1ng, the product of MDA heterozygous allele amplification equilibrium; DNA template is less than 0.5ng, the MDA product of STR profiles showed allelic imbalance or loss, and the amount of template DNA is low, allelic imbalance or loss phenomenon is more obvious as the template.DNA more than 0.05ng, the product of IPEP heterozygous allele amplification equilibrium; DNA template is less than 0.025ng, the IPEP product of STR profiles showed allelic imbalance or loss of.MDA products and IPEP products for STR types were not observed in non specific product peaks.
4. with better fidelity DNA polymerase and random primer dosage, established.0.1ng, improved IPEP method 0.05ng, 0.025ng, 0.01ng four group template of DNA by modified IPEP method amplified product concentration were higher than those of IPEP, there were significant differences (F=289.899, P < 0.001). Modified IPEP was amplified by 3ng 84ng products on the DNA template, the template DNA increased 160~1220 times the template.DNA was 0.025ng, the improved IPEP method can obtain complete and accurate genotyping results the template.DNA was 0.01ng, the average detected loci of modified IPEP was higher than the number of IPEP, some loci not detected by D7S820, D18S51, FGA. Compared with common products. The modified IPEP method for STR typing, no non specific product peaks and allelic imbalance or loss.
5. the improved IPEP method was used for the accurate typing of all loci in 0.025ng blood DNA, blood yarn DNA, and semen DNA.
6. improved IPEP method can significantly improve the sweat latent fingerprints DNA (t=5.423, P < 0.001), disposable toothbrush (t=6.835, P < 0.001 DNA) the number of loci detected, but the natural shedding hair DNA loci detected number is lower than that of the conventional detection method (t=3.249, P < 0.001). The modified IPEP method on the stones, the actual case in the cup, cigarette butts, collar and other samples the detection rate and the average number of detected loci were higher than that of the conventional detection method and detection of STR loci were not less than 9.
conclusion
The yield of 1.MDA is high, but the sensitivity is low. When the template quantity is below 1ng, the product sequence is poor, which affects the accuracy of subsequent STR typing. Therefore, MDA method is effective for retaining samples, but it is not suitable for trace DNA detection.
The 2.IPEP method has high sensitivity and good product sequence fidelity, and a good STR typing effect can be obtained for the genomic DNA of 0.05ng, but the yield is low.
3.DNA and random primer polymerase dosage has important effect on IPEP method. With better fidelity of DNA polymerase and random primer to a certain concentration, improve the amplification efficiency. Product sequence fidelity improved IPEP method based on the good, and the yield and sensitivity are further improved, improved trace DNA STR type effect.
4. the improved IPEP method is accurate and reliable for different biological samples, such as oral swab, blood, blood yarn, semen, etc., and shows that the improved IPEP method is stable and suitable for forensic biological examination.
5. improved IPEP method can significantly improve the micro skin cells, oral mucosal exfoliated cell samples of trace detection success rate and the average number of loci detected, the cumulative discrimination reached that level, to meet the requirements of forensic identification. But the improved IPEP method for poor natural hair off this type of degradation of short fragments the DNA sample results.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919

【引证文献】