Establishment Of Whole Genome Amplification Method And Its Application In Forensic Medicine

Background and ObjectivesEvidentiary items often contain insufficient quantity of DNA for routine forensic genetic analysis. These minute DNA samples are often undetected successfully by routine DNA analysis method or have no sufficient quantity of DNA for repeated analysis. So minute DNA analysis is a difficult task in forensic science. Since PCR-STR method was applied in forensic DNA analysis, some difficulties of minute DNA analysis have been resolved. However, several samples with insufficient quantity of DNA are still not detected successfully by this method, or the loci detected is too few to meet the need of individual identification. These samples DNA is called trace DNA or low copy number (LCN). It means less than 100pg of template DNA. In an attempt to resolve this difficulty, the use of increased cycle number has been suggested. However, this method may not always be efficient, allele drop and unexpected allele will be observed when the amount of template DNA is very low. However increasing cycle numbers cannot increase the sensitivity when cycle numbers are increased to some extent. Similarly, nested PCR has been suggested for LCN samples. But it is difficult to envision its use with forensic specimens due to multiple primer compatibility with multiple multiplex PCR systems. Whole genome amplification (WGA) has been considered as a fundamental method for forensic DNA analysis. WGA technique employs various random-sequence primers and low stringency annealing conditions to amplify large tracts of the genome, aimed to obtain sufficient quantities of template DNA from limited amount of genomic DNA.During the past decade, several WGA methods have been developed. Effective implementation of a robust WGA method has had tremendous impact on the fields involving genetic marker analysis, such as clinical genetics study. However, few studies have demonstrated the suitability of these WGA methods for forensic caseworke. Currently, there are some effective WGA methods such as improved primer extension preamplification (IPEP) and multiple displacement amplification (MDA). Both IPEP and MDA could have obtained good results for STR analysis. So this study focuses on the effect of MDA and IPEP for forensic DNA analysis, including their sensitivity, fidelity, STR coverage and applicability for forensic medicine.Methods1. DNA was extracted from 30 buccal swabs by standard phenolchloroform method and quantitated by real-time PCR. These samples DNA was made appropriate dilutions to obtain various amounts of DNA included the following: 1ng, 0.5ng, 0.1ng, 0.05ng, 0.025ng and 0.01ng. These samples were amplified by both MDA and IPEP. The concentration of MDA products and IPEP products was detected by real-time PCR and the amplification efficiency of the two WGA methods was evaluated. Besides, WGA products obtained by MDA and IPEP protocols were evaluated with AmpFLSTR^?Identifiler^?. Genetypes were detected by ABI3100 Genetic Analyzer. And the effects of the MDA products and IPEP products on STR analysis were compared.2. The reaction components of IPEP and their final concentration were optimized to establish modified IPEP. The yield and STR analysis effect of modified IPEP were evaluated. DNA originating from 30 blood samples, 30 dried bloodstains samples and 30 semen samples were detected to ensure that modified IPEP would perform consistently with various cell types.3. DNA samples originating from sweat latent fingerprints, toothbrushes once-used and telogen hairs were detected to evaluate STR analysis efficiencies of modified IPEP. And fruit-stones, water bottle, cigarette butts and clothes were detected to demonstrate the suitability of modified IPEPfor forensic casework specimens.Results 1. The products concentration of MDA was higher than IPEP and the difference is significant (F=3643.433,P<0.001). The quantification of the WGA products with starting g-DNA from 0.01ng to 1ng showed yields of 5.15ug～19.75ug for MDA products and 0.5ng⁶6ng for IPEP products. The template DNA was increased 5.15×10³ï½ž1.98×10⁶ and 25～310 times by MDA and IPEP respectively.2. There were significant differences in the number of detected loci of varying amount template DNA that was respectively amplified by routine forensic genetic analysis, MDA and IPEP (F=1033.297, P<0.001). When the template DNA was 1ng, all 16 loci were detected by routine forensic genetic analysis, MDA and IPEP. When the template DNA was 0.5ng, all 16 loci were detected by both routine forensic genetic analysis and IPEP, and more than 10 accurate loci were detected by MDA. When the template DNA was 0.1～0.01ng, the number of detected loci of MDA was more than routine forensic genetic analysis, and the number of detected loci of IPEP was more than MDA.3. When the template DNA was not less than 1ng, there was no allelic imbalance of heterozygotes in MDA products. When the template DNA was not more than 0.5ng, there was allelic imbalance of heterozygotes in MDA products. When the template DNA was more than 0.05ng, there was no allelic imbalance of heterozygotes in IPEP products. When the template DNA was less than 0.025ng, allelic imbalance of heterozygotes was observed in some loci of IPEP products. Non-specific peaks were not observed in both MDA products and IPEP products.4. The product concentration of modified IPEP was higher than IPEP and the difference was significant (F=289.899,P<0.001). The starting g-DNA from 0.01ng to 0.1ng showed yields of 3ng-84ng after modified IPEP. The template DNA was increased 160-1220 times by modified IPEP. When the template DNA was 0.025ng, all correct loci were detected by modified IPEP. When the template DNA was 0.01ng, the number of detected loci of modified IPEP was more than IPEP, but some loci weren't detected. The undetected loci were mainly observed in D7S820, D18S51 and FGA. Neither non-specific peaks nor allelic imbalance was observed in products of modified IPEP. 5. All specimences of 0.025ng DNA originating from blood, dried bloodstains and semen amplified by modified IPEP had obtained accurate genetypes of all detected loci.6. There were significant differences in the number of detected loci of fingerprint DNA, toothbrush DNA and hair DNA, which were respectively amplified by modified IPEP and by routine forensic genetic analysis (F=265.080, P<0.001). The numbers of detected loci of fingerprint and toothbrush by modified IPEP were more than those by routine forensic genetic analysis. The numbers of detected loci of hair DNA by modified IPEP were less than those by routine forensic genetic analysis. The numbers of detected loci of DNA origining from fruit-stones, water, cigarette butts and clothes amplified by modified IPEP were significantly higher than those by routine forensic genetic analysis, and the numbers of detected loci were not less than 9.Conclusion1.The yield of MDA is higher but the sensitivity is lower. When the template DNA is less than 1ng, the fidelity of MDA products is worse, which will influence accuratissime of the STR analysis. So MDA is useful for keeping samples DNA, but is not a good tool for trace DNA analysis.2.The sensitivity of IPEP is higher and the fidelity of IPEP products is well. Genomic DNA of more than 0.05ng amplified by IPEP show a good STR genotyping but its yield is lower.3. The Taq DNA polymerase and the amount of primers have obvious effect on IPEP. Using the enzyme of higher fidelity and increasing the final concentration of random primers can promote fidelity of modified IPEP products and amplification efficiency, and improve the STR genotyping of trace DNA.4. The modified IPEP products of DNA originating from buccal swabs, blood, dried bloodstains and semen used for STR analysis showed good effects comfirms the method is consistently with various cell types. 5.Modified IPEP can increase the number of detected STR loci of minute skin cells and buccal membrane cells and meet the need of individual identification in forensic medicine. But degraded DNA samples such as telogen hairs are not suitable for this method.