Comparative Study On DNA Extraction And Purification For Touch DNA And Its Application In Forensic Medicine

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 samplesDNA 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 samplesDNA 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 methodsThe 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.