| DNA evidence has become one of the most effective techniques to reveal the criminals, identified the suspects and one of the conviction and sentencing in forensic science. Fingerprints and other traces of evidence is often conscious destruction and damage due to anti-reconnaissance criminals increasing. DNA technology has been used widely in criminal case with the technology ripening and further understanding. DNA testing technology is also beginning to play an increasingly important role in multiple cases, especially theft cases, in addition to the major and difficult cases such as murder cases and rape cases. And cigarette butts, as a common trace samples, has been a favored samples by investigators because of simple extraction and packaging, and easy detection. Furthermore, cigarette butts samples can be found in 60% thefts scene according to the data of theft cases in 2008-2012 from PanYu branch of Guangzhou Public Security Bureau. Besides, in forensic work, more than 90% of the cases encountered in biological samples of exfoliated cells class Trace. And individual differences among the laboratory Personnel can not be ignored. How to reduce the burden of heavy manual operation and individual difference, improve the extraction rate of exfoliated cells from different sources has become a major breakthrough point in the development of DNA technology.Currently the technology of automation workstation coupling commercial bead kit has been applied in some Chinese laboratories in batch extraction of routine biological samples such as blood stein and tissues, which means the normalization and the automation has been accomplished in forensic detection. The large-scale batch workload automation platform for DNA extraction has been used in some cases, but manual and semi-automated extraction is still the major method for DNA extraction in most of the primary laboratories. We have initially established a method for suitable exfoliated cells batch-extracted with nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit kit system and used the method for batch-detection of cigarette butts in practical cases. AND the results showed that the method was reliable and effective, which can be applied in batch extraction of exfoliated cells by primary laboratories.1 The development of batch extraction methodology with M48 kit30 cigarette butts from volunteers, were used for the development of the DNA extraction with nucleic acid extraction workstation epMotion 5075 coupling MagAttract (?) DNA Mini M48 Kit, and the results was compared with that of manual-chelex-100 extraction method nucleic acid extraction workstation epMotion 5075 coupling IQTM, and nucleic acid extraction workstation epMotion 5075 coupling MagAttract (?) DNA Mini M48Kit, respectively.It was found that the complete DNA typing can be obtained with the process of batch extraction of the cigarette papers using nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit kit, whether in the different lysis systems,,as long as the cigarette paper can be completely immersed in Lysates. the complete DNA typing can be obtained if lysis time is more than 30 minutes. The lysis effect was not affected by whether the membrane’s sealed in the lysis process,,and cross-contamination between samples was not occurred in the lysis process.The concentration of DNA extraction with manual-chelex-100 extraction method, IQTM system extraction and M48 system extraction was 0.8307ng/μl,0.4875 ng/μl and 0.4990 ng/μl ug/mL, respectively. But the samples of the 30 cigarette butts extracting with three methods, which detected genetypes in more than 12 STR loci,was respectively 26,27 and 29。It was found that the M48 Kit system coupling nucleic acid extraction workstation epMotion (?) 5075 extract of cigarette butts off the cells better than hand-chelex-100 extraction and nucleic acid extraction workstation epMotion 5075 coupling DNA IQTM.2 The comparison of the batch-extraction effects in different cigarette butts samplesThe 600 cigarette butts of 6 different brands of Guangdong production collected from volunteers were batch; and 40 cigarette butts were randomly selected from 600 cigarette butts of 6 different brands of Guangdong production and collected from volunteers.The 40 cigarette butts were batch detected after placed for 1,7 and 10 weeks, and the extraction effects were compared. Furthermore the extraction procedure was optimized and batch extraction of trace exfoliated cells workstation extract system was further improved.The extraction rate was significant affected by the permeability of the carrier. The permeability of medium and top grade cigarette butts was poorer due to Impermeable processing. And when medium and top grade cigarette butts detected it was found the number of detected was 92 and 80 cases which genetypes were less than 12 STR locus with 150ul/s and 100ul/s transport velocity; all the samples were detected with ≥12 STR locus with 50ul/s transport velocity. While all the samples were detected with ≥12 STR locus with three transport velocity above mentioned when low grade of cigarette butts detected. Thus, for a less permeable carrier, it is possible to appropriately lower the speed of transfer Lysates to avoid or reduce the chance of clogging pipette tip.More than 12 STR locus was detected from the low and mid-range of cigarette and shuangxi Yiping cigarette. There were 8 cases that 9-12 STR locus was detected from the top grade Wu-Ye-Shen cigarette papers because of pigment overflow. To compare the average Peak height of D19S433, D3S1358, D8S1179, D6S1043 which’s the peak’s sizes between 101.68-190.83 and D7S820, D18S51, CSF1PO, D2S1338 which’s the peak’s sizes between 255.26-359.29,with the above-mentioned 30 cases. to compared with the other five brands,the Comparison value of the average Peak height between the lager and small peak was smallest of Wu-Ye-Shen cigarette papers It’s because of the Inhibitor in the process of PCR. to compared the average Peak height with the above-mentioned 30 WU-YE-SHEN cigarette between the result of the second and first washing, the Comparison value between the lager and small peak was bigger,but the average Peak height was smaller. So increasing the number of washing, it is possible to further reduce the concentration of inhibitor, but have a certain influence of the concentration of DNA extraction. So it’s necessary for us to design a suitable extraction procedure coupling the conditions of the sample.It is concluded that adjustment the extraction procedures according to the different biological sample is a better protocol to further reduce the impact of carrier on extraction.3 The comparison of the test effects of cigarette butts samples among three systemsDNA production was extracted from 100 cigarette butts provided by volunteers using nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit. And the genotyping consistency was studied with Sino-filers PowerPlex 21 AGCU EX22 STR amplification system.The results show that the gene typing of 15 STR locus with two methods, nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit and Sino-filers PowerPlex 21 AGCU EX22 STR amplification system, were exactly the same and no leakage or locus deletion occurred. Of the three different amplification systems, the peak height and peak area in the same STR locus were same on the whole. And there is no locus of PHR≤0.5 between peak heights of heterozygous and no obviously uneven amplification.The individual difference of DNA extraction caused by the different experimenters, samples and extraction methods is a serious problem in DNA detection. It is concluded that our method, using nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit, can avoid the problems and the method is particularly suitable for automated extraction of trace DNA extraction of contaminated samples.4 Case ApplicationsOur new developed extraction method was compared with the other two extraction systems in practica application. The skin exfoliated cells from 200 daily cases were extracted using nucleic acid extraction workstation epMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit; the samples from PanYu branch of Guangzhou Public Security Bureau were processed by the chelex-100 extraction since 2008 or Maxwell 16 nucleic acid purification instrument extraction since 2010. The results showed that the positive rate of the new method was consistent with that of chelex-100 extraction system, and the extraction efficacy and normalization was superior to that of Maxwell 16 nucleic acid purification instrument extraction system. The new developed method show a higher positive rate of batch extraction than chelex-100 extraction system.In summary, we have developed a method of EpMotion (?) 5075 coupling MagAttract (?) DNA Mini M48 Kit kit extraction system for cigarette butts batch extraction and trace sample extraction. The new method can be promoted in primary laboratories and will be conducive to normalization and automation in primary laboratories. And the feasibility of the new method has been further demonstrated in practical work.. |
PDF Full Text Request