【摘要】： 接觸DNA (touch DNA)檢驗是法醫(yī)DNA檢驗的難點和熱點之一。法醫(yī)鑒定中的接觸DNA檢材,是指含有人體皮膚粘膜的脫落細(xì)胞的檢材,包括含口腔脫落細(xì)胞的檢材、含體表脫落細(xì)胞的檢材、人體排泄物或分泌物等。凡是與人體皮膚粘膜接觸過的物品,都可能留下接觸者的脫落細(xì)胞。這些留有脫落細(xì)胞的物證可以存在于犯罪現(xiàn)場,也可存在于犯罪嫌疑人及被害對象的衣物、用具等客體上。隨著犯罪分子反偵察手段的日益狡猾,明顯的生物物證(精斑、血斑、毛發(fā)、人體組織等)常被有目的地銷毀和破壞,而來自人體的代謝脫落細(xì)胞由于微小痕量,常被忽略,如遺留在煙蒂、飲料罐、果核、口香糖上的口腔脫落上皮細(xì)胞,遺留在手套、衣物、帽子、指紋上的皮膚脫落上皮細(xì)胞,等等。對于這些潛在的微量生物物證,一旦成功地獲得了關(guān)鍵物證的DNA分型,往往就能夠成為案件偵破的關(guān)鍵線索,并為案件定性及法庭量刑提供科學(xué)證據(jù)。 如何從微量接觸檢材中提取到足夠的DNA以滿足法醫(yī)學(xué)檢驗的需要,選擇合適的提取方法非常關(guān)鍵,是法醫(yī)物證檢驗工作者最關(guān)注的難題之一。接觸DNA檢材與常見的生物檢材如血痕、精斑相比,屬于疑難生物檢材。首先,肉眼觀察很難判斷接觸細(xì)胞在載體上的具體部位,盲目提取難以獲得所需靶細(xì)胞；其次,擦拭面積太大會降低靶DNA的濃度,并帶來外源物質(zhì)的污染和干擾；第三,接觸DNA檢材含有的DNA往往是微量的,屬于低拷貝數(shù)目(Low copy number,LCN)模板,即“DNA含量少于l00pg,相當(dāng)于15個雙倍體或30個單倍體細(xì)胞的生物樣本”。因此接觸DNA檢驗不同于常規(guī)生物檢材的DNA檢驗,更需系統(tǒng)研究。 目前關(guān)于接觸DNA提取純化方法的文獻(xiàn)很多,概括起來有Chelex法、磁珠法、有機(jī)溶劑法(有機(jī)法)、過柱法、硅珠法、硅膠膜法等。但多為針對某種方法對某種類型接觸DNA的提取或案例報道,對不同提取純化方法獲得的現(xiàn)場不同類型的接觸DNA的質(zhì)與量的橫向比較缺乏系統(tǒng)研究。本研究采用Quantifiler人類DNA定量試劑盒(AB, USA)實時定量技術(shù)對目前常用的提取純化方法包括Chelex法、磁珠提取法(DNA IQ磁珠法、EQ國產(chǎn)磁珠法)、有機(jī)溶劑提取法、Microcon過柱法提取的接觸DNA進(jìn)行定量,同時用Identifiler或Sinofiler復(fù)合擴(kuò)增系統(tǒng)(AB,USA)擴(kuò)增,在AB 3130遺傳分析儀上進(jìn)行STR (short tandem repeat,短串聯(lián)重復(fù))分型。綜合DNA定量和STR分型結(jié)果,對各種提取純化方法獲得的DNA的質(zhì)和量進(jìn)行評估和系統(tǒng)比較,以建立有效的接觸DNA提取純化方法。 1. Chelex法提取接觸DNA的實時定量研究 對使用后放置1個月以內(nèi)的30個飲料瓶口或吸管、30個礦泉水瓶(杯)口分別采用不洗滌直接加Chelex-100提取與洗滌+Chelex-100的方法提取DNA,通過PCR定量和STR檢測,比較2種處理方式獲得的DNA平均含量、IPC Ct值、STR檢驗成功率；對使用后放置1個月以內(nèi)的10個新鮮飲料瓶口或吸管,使用后分別放置1個月和放置2個月的20把牙刷、20枚煙蒂,分別采用洗滌+Chelex-100和洗滌+Chelex-100+蛋白酶K(PK)的方法提取DNA,通過PCR定量和STR檢測,比較2種處理方式獲得的DNA平均含量、IPC Ct值、STR檢驗成功率。研究結(jié)果表明,Chelex-100提取前加浸泡洗滌的步驟,可去除部分可溶性雜質(zhì),有利于提高獲得的DNA量和STR檢驗成功率,同時降低反映PCR抑制物存在的IPC Ct值。室溫放置1個月左右的新鮮接觸DNA檢材用Chelex法提取時,是否加蛋白酶K對提取的DNA量和STR檢驗成功率無顯著影響；室溫放置2個月以上的接觸DNA檢材用Chelex法提取時,加蛋白酶K消化后獲得的DNA量和STR檢驗成功率均高于不加蛋白酶K的樣品,差異有顯著性意義。本研究對180份10種實際案件的接觸DNA檢材用Chelex法提取,煙蒂獲得的平均DNA含量最高達(dá)175.85 ng,剃須刀較低為7.80 ng。除煙蒂、口香糖采用常規(guī)體系提取外,其它檢材采用小體系提取,平均DNA濃度均在0.2ng/μl以上,采用2μll模板復(fù)合擴(kuò)增,除衣服、剃須刀的STR分型成功率較低為50%外,其它檢材的STR分型成功率均在60%以上。 2.磁珠法提取接觸DNA的實時定量研究 對煙蒂、牙刷、手套等3種接觸DNA檢材各10個分別采用95℃裂解液直接裂解、70℃裂解液直接裂解和預(yù)消化(TNE+SDS+PK)等3種前處理方式后用DNA IQ磁珠(Promega, USA)提取DNA,測定并比較三種前處理方式后用DNAIQ磁珠法提取獲得的DNA平均含量和IPCCt值。研究結(jié)果表明,接觸DNA檢材采用先加TNE、SDS、PK等消化,然后再用磁珠純化的方法獲得的DNA量高于單純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觸DNA檢材的STR檢驗成功率。但該處理方法操作相對繁瑣,耗時長,除了污染嚴(yán)重的微量接觸DNA檢材外,大部分接觸DNA檢材單純采用直接裂解法就可以獲得足以進(jìn)行STR分析的DNA。裂解溫度方面,95℃裂解與70℃裂解的DNA獲得量無顯著性差異。對151份8種實際案件的接觸DNA檢材采用95℃直接裂解的DNAIQ磁珠法在MaxwellTM 16自動儀上提取DNA。結(jié)果表明除果核平均DNA獲得量為9.51ng以外,其它7種接觸檢材的平均DNA獲得量均大于10ng。采用30μl的小洗脫體積,平均DNA濃度可達(dá)0.3ng/μl,采用8μl體系3μl模板擴(kuò)增,檢驗成功率最低的果核也達(dá)60%。EQ國產(chǎn)磁珠法與DNA IQ磁珠法相比,不僅價格便宜,還少了1個裂解液洗滌和1個洗滌液洗滌的步驟,操作更為簡單,但對接觸DNA的提取效率無顯著性差異。對138份6種實際案件的接觸DNA檢材在自動化工作站經(jīng)EQ國產(chǎn)磁珠法提取DNA,采用8μl體系3μl模板擴(kuò)增,除對飲料瓶口的檢驗成功率較低,為60%外,對煙蒂、口香糖、手套等接觸DNA檢材的檢驗成功率也能達(dá)到70%以上。因此也適合接觸DNA的提取。 3.5種接觸DNA檢材提取純化方法的比較 將稀釋為lOng、100ng的標(biāo)準(zhǔn)品DNA,分別采用Chelex法、DNA IQ磁珠法、EQ國產(chǎn)磁珠法、有機(jī)法、Microcon過柱法等5種DNA提取純化方法處理,然后進(jìn)行PCR定量,比較5種DNA提取純化方法對標(biāo)準(zhǔn)品DNA的回收率。對煙蒂和牙刷2種常見的接觸DNA檢材分別采用Chelex法、95℃直接裂解的DNA IQ磁珠法和EQ國產(chǎn)磁珠法提取DNA,通過PCR定量和STR檢測分析,比較3種提取方法提取的DNA平均含量、IPC Ct值和STR檢驗效果；對30例污染嚴(yán)重的接觸DNA檢材,先用TNE、SDS、PK消化,然后將消化液平均分成2份,1份用DNA IQ磁珠法純化,1份用有機(jī)法抽提,通過PCR定量和STR檢測分析,比較磁珠法與有機(jī)法對接觸DNA的純化效果；將Chelex法提取的15例5種接觸DNA溶液用Microcon100超濾柱進(jìn)行純化濃縮,通過PCR定量和STR檢測分析,比較Microcon過柱法濃縮前后的濃度、體積、IP Ct值及回收率。結(jié)果表明,Chelex法對DNA的提取無損失,磁珠法、有機(jī)法、Microcon過柱法對DNA的提取純化均有不同程度的損失。5種提取純化方法對DNA的回收率從高到低大致為Chelex法Microcon過柱法DNA IQ磁珠法EQ國產(chǎn)磁珠法有機(jī)法。Chelex法提取DNA,操作簡單快速,整個提取過程均在一個離心管中進(jìn)行,避免了DNA在提取過程中的損耗,對污染輕、雜質(zhì)少的接觸DNA檢材,用Chelex法提取最為方便快捷。但Chelex法不能去除DNA溶液中的雜質(zhì)和降解的DNA碎片,因此,Chelex法提取的陳舊接觸檢材的DNA進(jìn)行STR擴(kuò)增時,即使模板量在試劑盒推薦的范圍內(nèi),也容易出現(xiàn)等位基因擴(kuò)增不平衡的現(xiàn)象。磁珠法與Chelex法相比,雖然提取過程中會造成DNA的損失,但提取的DNA純度高,能有效去除100bp以下的DNA小片段和樣品中的色素、蛋白質(zhì)等雜質(zhì),因此,STR擴(kuò)增時峰高比較均衡,等位基因擴(kuò)增不平衡的現(xiàn)象不明顯。磁珠法與有機(jī)法對接觸DNA的回收量和IPC Ct值雖然無顯著性差異,但磁珠法避免了有機(jī)溶劑對操作者的潛在危害,操作簡單,耗時短,因此,更適合微量、污染檢材的DNA提取及自動化操作。Microcon過柱法操作簡單,但對接觸DNA的回收效果不一,100μl的起始體積,過柱濃縮后的體積在8-25μl之間,平均為15μl。過柱后,DNA濃度平均約增加5倍,對DNA的回收率平均為61%,但反映PCR抑制物存在的IPC Ct值無顯著降低。同時,Microcon過柱法的成本也高于磁珠法。因此,Microcon過柱法對接觸DNA的提取純化應(yīng)用有限,只適合污染輕的接觸DNA的純化濃縮。 本研究通過對5種常見的提取純化方法獲得的DNA的質(zhì)和量進(jìn)行系統(tǒng)比較和評估,為規(guī)范法醫(yī)檢驗中接觸DNA檢材的提取送檢以及法醫(yī)工作者如何根據(jù)檢材、實驗室設(shè)備等情況選擇合適有效的接觸DNA提取純化方法提供了科學(xué)依據(jù),有利于提高接觸DNA檢材的檢驗成功率,充分發(fā)揮接觸DNA檢材的證據(jù)價值。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：D919.4

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 曲春冰;張國翔;袁家龍;;KingFisher磁珠純化儀在提取脫落細(xì)胞檢材DNA上的應(yīng)用[J];廣東公安科技;2011年01期

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本文編號：2178241