| Objective: Gender information is one of the most important information in the forensic casework and archaeological research. Usually if the corpse is unbroken, forensic workers can make a determination by morphological analysis. However, in some disasters of large group, when the integrity of the human skeleton is destroyed, or bone hypoplasia as a child lead to identification by morphological analysis difficulty, using molecular biological methods to indentify gender becomes particularly important. At present, the commonly used DNA techniques in gender identification include Y chromosome-specific probes, Zin-finger protein gene (ZFY/ZFX) and amelogenin gene test, among which amelogenin test is the most widely applied. Since 1991 amelogenin gene had been sequenced by Nakahori, Nakahori (1991), Akane (1991), Bailey (1992), Sullivan (1993) had used PCR method to amplify the single copy X-Y homologous regions of amelogenin gene by different primers in sex determination. The method made by Sullivan with PCR products being 106bp and 112bp, has been widely used for gender identification, which was applied by most of the forensic personal identification kits as a sex identification method. However, these existing technologies can not fully meet the needs of a number of difficult cases, and usually can not get clear typing results for highly-degraded samples or ancient DNA, or can lead to invalid amplification result from amelogenin sequence mutation and sex chromosome variation. To solve these problems, this study established a new gender DNA analysis methods, namely, a 45bp stretch of amelogenin gene was chosen for analysis by pyrosequencing. At the same time, a series of validation experiments, such as the sensitivity, accuracy and species specificity, degradation models, bone samples, were performed for the novel method, with a view to provide a reference for sex determination in some difficult cases.Methods:⑴The nucleotide sequences of AMELX and AMELY were acquired from Genebank. According to the blast results, a fragment including three single nucleotide polymorphism loci and an indel motif was chosen to be as the target sequence. Then a pair of amplification primers and sequencing primer were designed by primer design software, such as primer5, Oligo6 and so on. The downstream primer was labeled with biotin. The expected sequencing model was established by Pyrosequencing software. The concentration of primer, Mg2+, Taq enzyme and the annealing temperature as well as the number of cycles were optimized to get the best PCR condition.Then, the PCR product was sequenced by Pyrosequencing. 100 randomly chosen DNA samples of healthy donors of known sex (50 from males, 50 from famales) were typed to verify the reliability and accuracy of this method.⑵Two DNA samples of men and women were quantified and diluted to 1ng, 0.5ng, 0.25ng, 0.125, and 0.0625ng to test the sensitivity. The method was been applied to test DNA samples of monkey, dog, rabbit, pig, cow, fish, and chicken, as well as Escherichia coli, Enterococcus faecalis, Candida albicans, to verify the species specificity. The blood was laid outside for 26 weeks from June to November to model degraded samples, and artificial degraded DNA was prepared by digesting with DNase I, which were analyzed by the pyrosequencing method and commercial AmpF / STR IdentifilerTM kit to compared the success rate of the two methods. In addition, 6 bone samples were analyzed using this method to identify gender.Results:⑴A method of short PCR products of amelogenin gene tested by pyrosequencing was successfully developed for gender identification. The 45-bp PCR products included primers (40bp) and target sequence (5bp) consisting of 3 point mutations and an indel mutation. The pyrosequencing graph was clear and easy to be typed. 100 DNA samples of known sex were typed with the above method and all of which got clear typing graphs and correct results, indicating the method was accurate and reliable.⑵ Evaluation of forensic application:①The sensitivity of the method was 1ng template DNA.②The study of species-specificity showed that among all of the detected non-primate animals (dog, rabbit, cow, fish, pig,chicken), primate animals (monkey) and common laboratory strains (Candida albicans, enterococcus , Escherichia coli), no specific peak was detected in all of the species except for monkey, in which the pyrosequencing graph was different from that of humans.③For the eight samples of highly degraded DNA extracted from blood laied outside for 26 weeks, all of them were correctly typed by the new method, whereas only six of them were acquired results by IdentifilerTM kit, suggesting the new method had a higher successful rate for analysis of degraded samples than the conventional gender identification method.④For the artificial degraded DNA digested by DNase I for 8 different times of 5min, 10min, 20min, 25min, 30min, 40min and 45min, all of them were typed correctly and clearly by the new method except 45min. However, when testing with IdentifilerTM kit, there were lower peaks of amelogenin gene product only for the DNA digested for 5min and 10min, and other degraded DNA digested for longer time were failed to be typed.⑤Among the 6 bones analyzed, except that the teeth had a failure typing due to less material, the remaining five were typed clearly.Conclusions: A gender identification method was constructed, which tested amelogenin gene by pyrosequencing with amplification product being only 45bp. This method was easily-operated, quick, low cost, with high-throughput, high sensitivity of 1ng DNA, and human species specificity. It was also accurate and stable because of target sequence including 3 point mutations and an indel mutation. Especially the new method had a higher successful rate for analysis of highly degraded samples than the conventional gender identification method. Based on these advantages, this method can be recommended to identify gender of highly-degraded samples or ancient DNA samples, and investigation of large scale samples.
|
PDF Full Text Request