| Background and ObjectivesEvidentiary items often contain insufficient quantity of DNA for routine forensicgenetic analysis. These minute DNA samples are often undetected successfully byroutine DNA analysis method or have no sufficient quantity of DNA for repeatedanalysis. So minute DNA analysis is a difficult task in forensic science. SincePCR-STR method was applied in forensic DNA analysis, some difficulties of minuteDNA analysis have been resolved. However, several samples with insufficientquantity of DNA are still not detected successfully by this method, or the locidetected is too few to meet the need of individual identification. These samples DNAis called trace DNA or low copy number (LCN). It means less than 100 pg oftemplate DNA. In an attempt to resolve this difficulty, the use of increased cyclenumber has been suggested. However, this method may not always be efficient, alleledrop and unexpected allele will be observed when the amount of template DNA isvery low. And increasing cycle numbers can not increase the sensitivity when cyclenumbers are increased to some extent. Similarly, nested PCR has been suggested forLCN samples. But it is difficult to envision its use with forensic specimens due tomultiple primer compatibility with multiple multiplex PCR systems. Whole genome amplification (WGA) has been considered as a fundamental method for forensic DNAanalysis. WGA technique employs various random-sequence primers and lowstringency annealing conditions to amplify large tracts of the genome, aimed to obtainsufficient quantities of template DNA from limited amount of genomic DNA.During the past decade, several WGA methods have been developed. Effectiveimplementation of a robust WGA method have had tremendous impact on the fieldsinvolving genetic marker analysis, such as clinical genetics study. However, fewstudies have demonstrated the suitability of these WGA methods for forensiccasework. Currently, there are some effective WGA methods, such as improvedprimer extension preamplification (IPEP) and multiple displacement ampliWcation(MDA). And both IPEP and MDA are good for STR analysis. So we focus on thestudy of the effect of MDA and 1PEP for forensic DNA analysis, including theirsensitivity, fidelity, STR coverage and applicability for forensic medicine.Methods1. DNA was extracted from 30 buccal swabs using a standard phenol-chloroformmethod and quantitated by real-time PCR. These samples DNA was made appropriatedilutions to obtain various amounts of DNA. The DNA amounts included thefollowing: 1ng, 0.5ng, 0.1ng, 0.05ng, 0.025ng and 0.01ng. These samples wereamplified by both MDA and IPEP. The concentration of MDA products and IPEPproducts was detected by real-time PCR and the amplification efficiency of the twoWGA methods was evaluated. Besides, WGA products obtained by using MDA andIPEP protocols were evaluated with AmpFLSTR? Identifiler?. Genetypes weredetected using ABI 3100 Genetic Analyzer. And the effect of the MDA products andIPEP products on STR analysis was compared.2. The reaction components of IPEP and their final concentration were optimizedto establish modified IPEP. The yield and STR analysis effect of modified IPEP were evaluated. DNA originating from 30 blood samples, 30 dried bloodstains samples and30 semen samples were detected to ensure that modified IPEP would performconsistently with various cell types.3. DNA originating from sweat latent fingerprints, toothbrushes used once andtelogen hairs was detected to evaluate STR analysis efficiencies of modified IPEP forthese samples. And fruit-stones, water bottle, cigarette butts and clothes were detectedto demonstrate the suitability of modified IPEP for forensic casework specimens.Results1. The products concentration of MDA is higher than IPEP and the difference issignificant (F=3643.433, P＜0.001). The quantification of the WGA products withstarting g-DNA from 0.0Ing to lng showed yields of 5.15μg-19.75μg for MDAproducts and 0.5ng-66ng for IPEP products. The template DNA was increased5.15×103-1.98×106 fold by MDA and 25-310 fold by IPEP.2. There is significant difference in the number of detected loci of varyingamount template DNA that was respectively amplified by routine forensic geneticanalysis, MDA and IPEP (F=1033.297, P＜0.001). When the template DNA was 1ng,16 loci were all detected by routine forensic genetic analysis, MDA and IPEP. Whenthe template DNA was 0.5ng, all 16 loci were detected by both routine forensicgenetic analysis and IPEP, and more than 10 accurate loci were detected by MDA.When the template DNA was 0.1ng-0.01ng, the number of detected loci of MDAwas more than routine forensic genetic analysis, and the number of detected loci ofIPEP was more than MDA.3. When the template DNA was not less than 1ng, there was no allelicimbalance of heterozygotes in MDA products. When the template DNA was notmore than 0.5ng, there was allelic imbalance of heterozygotes in MDA products.When the template DNA was more than 0.05ng, there is 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. Nonon-specific peaks were observed in both MDA products and IPEP products.4. The products concentration of modified IPEP is higher than IPEP and thedifference is significant (F=289.899, P＜0.001). The starting g-DNA from 0.01ng to0.1ng showed yields of 3ng-84ng after modified IPEP. The template DNA wasincreased 160-1220 fold by modified IPEP. When the template DNA was 0.025ng,all correct loci were detected by modified IPEP. When the template DNA was0.01 ng, the number of detected loci of modified IPEP was more than IPEP. But someloci weren't detected. The undetected loci were mainly observed in D7S820,D18S51 and FGA. Neither non-specific peaks nor allelic imbalance was observed inproducts of modified IPEP.5. 0.025ng DNA originating from blood, dried bloodstains and semen amplifiedby modified IPEP all obtained accurate genetypes of all detected loci.6. There was significant difference in the number of detected loci of fingerprintDNA (t=5.423, P＜0.001), toothbrush DNA (t=6.835, P＜0.001) and hair DNA (t=3.249, P＜0.001), which were respectively amplified by modified IPEP and byroutine forensic genetic analysis. The number of detected loci of fingerprint andtoothbrush by modified IPEP was more than by routine forensic genetic analysis. Thenumber of detected loci of hair DNA by modified IPEP was less than by routineforensic genetic analysis. The number of detected loci of DNA origining fromfruit-stones, water, cigarette butts and clothes amplified by modified IPEP wassignificantly higher than by routine forensic genetic analysis. And the number ofdetected loci was not less than 9.Conclusion1. The yield of MDA is high but the sensitivity is low. When the template DNA is less than lng, the fidelity of MDA pi-oducts is bad, which will influenceaccuratissime of the STR analysis. So MDA may be not a good tool for trace DNAanalysis.2. The sensitivity of IPEP is high and the fidelity of IPEP products is good.Genomic DNA Of more than 0.05ng amplified by IPEP showed good STRgenotyping. But its yield is low.3. The Taq DNA polymerase and the amount of primers have conspicuous effecton IPEP. Modified IPEP was established by utilizing a kind of enzyme of higherfidelity and increasing the final concentration of random primers. The fidelity ofmodified IPEP products is good. And modified IPEP has raised amplificationefficiency and improved 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. It comfirmsmodified IPEP performs consistently with various cell types.5. Modified IPEP can increase the number of detected STR loci of minute skincells and buccal membrane cells, to meet the need of individual identification inforensic medicine. But degraded DNA samples such as telogen hairs are not suitablefor this method.
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