發(fā)布時(shí)間：2018-04-16 07:29

  本文選題：全基因組擴(kuò)增 + 多重置換擴(kuò)增反應(yīng)�。� 參考：《蘭州大學(xué)》2008年碩士論文

【摘要】： 研究背景與目的 在法醫(yī)學(xué)檢案實(shí)踐中,常遇到樣本DNA含量極其有限,以致DNA分型無(wú)法完成或不能滿足重復(fù)檢測(cè)的需要。因此,對(duì)微量模板DNA分析便成為法醫(yī)學(xué)DNA檢驗(yàn)的一個(gè)難題。以PCR-STR為代表的遺傳標(biāo)記分析技術(shù)雖然一定程度上解決了法醫(yī)學(xué)微量檢材的難題,但一些檢材仍因DNA含量極其有限而無(wú)法檢測(cè)成功,或僅檢出少數(shù)基因座而無(wú)法使累計(jì)鑒別能力達(dá)到認(rèn)定水平。這類檢材被稱為痕量DNA,也稱低拷貝模板(low copy number,LCN),Gill等將其定義為低于100pg的模板DNA。為解決這一難題,有研究用增加PCR循環(huán)數(shù),但模板DNA量太低時(shí)易產(chǎn)生等位基因丟失或非特異帶,且循環(huán)數(shù)增加到一定程度后再增加循環(huán)數(shù)也不會(huì)顯著增加靈敏度;另外,有人試圖用巢式PCR,但巢式PCR技術(shù)僅限于單一位點(diǎn)分析,無(wú)法提高目前法醫(yī)學(xué)中常規(guī)DNA檢測(cè)所用的多位點(diǎn)復(fù)合擴(kuò)增所需的模板數(shù)量。全基因組擴(kuò)增(Whole Genome Amplification,WGA)是一種能從有限的DNA樣品獲得充足的DNA量供后續(xù)分析的技術(shù),其基本思路是通過(guò)對(duì)微量組織甚至單個(gè)細(xì)胞的整個(gè)基因組DNA擴(kuò)增,大幅度增加DNA的總量。因此被認(rèn)為是目前可以解決上述難題的一種有效方法。 WGA技術(shù)經(jīng)10余年探索,在方法上已逐步發(fā)展和不斷完善起來(lái),已被廣泛用于疾病基因研究等領(lǐng)域,并取得了良好的效果。但在法醫(yī)學(xué)方面的應(yīng)用很少。在目前常用的幾種WGA方法中,改良擴(kuò)增前引物延伸方法(Improved primerextension preamplification,IPEP)和多重置換擴(kuò)增方法(Multiple displacementamplification,MDA)對(duì)STR基因座分型顯示出較好的效果。故本研究探討了這兩種方法用于法醫(yī)學(xué)微量檢材的效果,包括對(duì)其靈敏度、擴(kuò)增忠實(shí)性、能否用于法醫(yī)學(xué)常用STR基因座及常用檢材等進(jìn)行系統(tǒng)性研究,從而對(duì)其用于法醫(yī)學(xué)實(shí)踐的可行性進(jìn)行評(píng)價(jià),并建立穩(wěn)定可行的技術(shù)方案。 方法 1.對(duì)30例口腔拭子樣品,采用酚-氯仿法提取DNA,實(shí)時(shí)熒光定量PCR技術(shù)定量,并分別稀釋成1ng、0.5ng、0.1ng、0.05ng、0.025ng、0.01ng六種模板量DNA,然后進(jìn)行MDA反應(yīng)和IPEP反應(yīng)。采用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)MDA方法和IPEP方法的產(chǎn)物濃度,比較兩種方法的擴(kuò)增效率。分別以MDA產(chǎn)物和IPEP產(chǎn)物為模板DNA,用AmpFLSTR~(?)Identifiler~(?)試劑盒擴(kuò)增,ABI3100基因分型儀檢測(cè)基因型,比較MDA產(chǎn)物和IPEP產(chǎn)物用于STR分型的效果。 2.對(duì)IPEP方法反應(yīng)體系的成分和終濃度進(jìn)一步優(yōu)化,建立改良IPEP方法,并檢測(cè)其擴(kuò)增效率和STR分型效果。將改良IPEP方法用于30例血液、30例血紗、30例精液等三類法醫(yī)學(xué)常見(jiàn)生物檢材進(jìn)行檢測(cè),觀察改良IPEP方法的可重復(fù)性和對(duì)法醫(yī)學(xué)常見(jiàn)生物檢材的適用性。 3.將改良IPEP方法用于20例汗?jié)撝赣　?0例一次性牙刷、20例自然脫落頭發(fā)等三種法醫(yī)學(xué)常見(jiàn)疑難微量檢材的模擬案例,觀察改良IPEP方法對(duì)上述檢材的STR分型效果;用于現(xiàn)場(chǎng)果核10例、礦泉水瓶10例、煙蒂10例、衣領(lǐng)10例等實(shí)際案例,觀察改良IPEP方法的實(shí)際案件檢驗(yàn)?zāi)芰Α?結(jié)果 1.MDA法產(chǎn)量為5.15ug～19.75ug,可對(duì)模板DNA增加5.15×10~3～1.98×10~6倍:IPEP法產(chǎn)量為0.5ng～66ng,可對(duì)模板DNA增加25～310倍。MDA法產(chǎn)物濃度高于IPEP法,差異有顯著性意義(F=3643.433,P＜0.001)。 2.常規(guī)檢測(cè)法、MDA法、IPEP法的基因座檢出數(shù)有顯著性差異(F=1033.297,P＜0.001)。當(dāng)DNA模板量為1ng時(shí),常規(guī)檢測(cè)方法、MDA方法、IPEP方法均可獲得復(fù)合擴(kuò)增系統(tǒng)所有基因座的準(zhǔn)確分型結(jié)果;當(dāng)DNA模板量為0.5ng時(shí),常規(guī)檢測(cè)方法和IPEP方法均獲得所有基因座的準(zhǔn)確分型結(jié)果,MDA方法獲得10個(gè)以上基因座的準(zhǔn)確分型結(jié)果;當(dāng)DNA模板量為0.1ng～0.01ng時(shí),MDA方法基因座檢出數(shù)高于常規(guī)檢測(cè)方法,IPEP方法基因座檢出數(shù)高于MDA方法。 3.DNA模板量≥1ng,其MDA產(chǎn)物雜合子基因座的等位基因擴(kuò)增均衡;DNA模板量≤0.5ng,其MDA產(chǎn)物的STR分型圖譜可見(jiàn)等位基因不平衡或丟失現(xiàn)象,且模板DNA量越低,等位基因不平衡或丟失現(xiàn)象越明顯。DNA模板量≥0.05ng,其IPEP產(chǎn)物雜合子基因座的等位基因擴(kuò)增均衡;DNA模板量≤0.025ng,其IPEP產(chǎn)物的STR分型圖譜可見(jiàn)等位基因不平衡或丟失。MDA產(chǎn)物和IPEP產(chǎn)物用于STR分型時(shí)均未觀察到非特異產(chǎn)物峰。 4.采用保真性更好的DNA聚合酶和增加隨機(jī)引物用量,建立了改良IPEP方法。0.1ng、0.05ng、0.025ng、0.01ng四組模板量DNA經(jīng)改良IPEP法擴(kuò)增所得產(chǎn)物濃度均高于IPEP法,差異有顯著性(F=289.899,P＜0.001)。改良IPEP法對(duì)上述模板DNA擴(kuò)增獲得3ng～84ng產(chǎn)物,可使模板DNA增加160～1220倍。DNA模板量為0.025ng時(shí),改良IPEP法可獲得完整準(zhǔn)確的分型結(jié)果。DNA模板量為0.01ng時(shí),改良IPEP法的平均基因座檢出數(shù)高于IPEP法,部分基因座仍未檢出,以D7S820、D18S51、FGA等較常見(jiàn)。改良IPEP方法的產(chǎn)物用于STR分型時(shí),未見(jiàn)非特異產(chǎn)物峰及等位基因不平衡或丟失。 5.改良IPEP方法對(duì)0.025ng血液DNA、血紗DNA、精液DNA檢測(cè)均獲得所有基因座的準(zhǔn)確分型結(jié)果。 6.改良IPEP方法可顯著提高汗?jié)撝赣NA(t=5.423,P＜0.001)、一次性牙刷DNA(t=6.835,P＜0.001)的基因座檢出數(shù),但對(duì)自然脫落毛發(fā)DNA的基因座檢出數(shù)低于常規(guī)檢測(cè)法(t=3.249,P＜0.001)。改良IPEP方法對(duì)實(shí)際案件中果核、杯口、煙蒂、衣領(lǐng)等檢材的檢測(cè)成功率和平均基因座檢出數(shù)均高于常規(guī)檢測(cè)方法,且檢出STR基因座數(shù)均不少于9個(gè)。 結(jié)論 1.MDA法產(chǎn)量高,但靈敏度低,當(dāng)模板量低于1ng時(shí),產(chǎn)物序列保真性差,影響后續(xù)STR分型的準(zhǔn)確性。因此,MDA法對(duì)于保留樣本有效,但不適用于痕量DNA檢測(cè)。 2.IPEP法靈敏度高,產(chǎn)物序列保真性好,對(duì)0.05ng的基因組DNA可獲得良好的STR分型效果,但產(chǎn)量低。 3.DNA聚合酶和隨機(jī)引物用量對(duì)IPEP方法有重要影響。采用保真性更好的DNA聚合酶和增加隨機(jī)引物至一定終濃度,提高了擴(kuò)增效率。在此基礎(chǔ)上建立的改良IPEP方法的產(chǎn)物序列保真性好,且產(chǎn)量和靈敏度都進(jìn)一步提高,改善了痕量DNA的STR分型效果。 4.改良IPEP方法對(duì)于口腔拭子、血液、血紗、精液等不同生物性檢材、不同狀態(tài)的同種檢材均獲得準(zhǔn)確可靠的結(jié)果,表明了改良IPEP方法的穩(wěn)定性好,適用于法醫(yī)學(xué)常見(jiàn)生物性檢材。 5.改良IPEP方法可顯著提高微量皮膚脫落細(xì)胞、口腔粘膜脫落細(xì)胞等痕量檢材的檢測(cè)成功率和平均基因座檢出數(shù),可使累計(jì)鑒別能力達(dá)到認(rèn)定水平,以滿足法醫(yī)學(xué)個(gè)體識(shí)別要求。但改良IPEP方法對(duì)自然脫落毛發(fā)這類降解的短片段DNA樣品效果不佳。
[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.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：D919

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 焦偉;劉斐;譚毅;;人短串聯(lián)重復(fù)序列相關(guān)技術(shù)及其在法醫(yī)學(xué)中的應(yīng)用研究進(jìn)展[J];中國(guó)臨床新醫(yī)學(xué);2011年11期

相關(guān)博士學(xué)位論文 前1條

1 彭薇;仿刺參（Apostichopus japonicus）微衛(wèi)星標(biāo)記的開(kāi)發(fā)與應(yīng)用[D];中國(guó)海洋大學(xué);2011年

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本文編號(hào)：1757857