One of the major problems of forensic genetics is the difficulty in analysis of degraded and low template (DaLT) DNA. The availability of only short and chemically modified DNA fragments results in lack of amplification of some DNA loci. Consequently, allele and locus dropouts disturb proper analysis of biological traces.1
One of the possibilities enabling recovery of DaLT DNA is applying a pre–polymerase chain reaction (PCR) whole-genome amplification (WGA) technique. Recently, several WGA methods and their modifications have been described, based in general on 2 different tactics using PCR or isothermal amplification. These use various techniques to increase the number of starting templates before any downstream analysis.2–10 Recently, we have described a comparison of 7 WGA methods in terms of their efficiency and usefulness in autosomal short tandem repeat (STR) loci analysis (SGMPlus; Life Technologies, Foster City, Calif), using degraded and nondegraded DNA.11 Here we demonstrate the results of Yfiler loci analysis after preamplification with these methods.
The value of the Y chromosome in forensics is that it is found only in males, which is especially important in cases where autosomal tests are limited by evidence, such as high level of female DNA in the presence of minor amounts of male DNA. Also, in archeological or historical cases, where male remains are revealed, Y-chromosome analysis is useful in determining the identity of the remains, by comparing Y-chromosome loci to living descendants. Especially in that kind of cases, DNA may be heavily degraded, and WGA methods may be applicable. Y-chromosome STR and single-nucleotide polymorphism (SNP) analysis is also performed at evolution, migration, and clinical research, as well as in analysis of formalin-fixed paraffin-embedded tissues in genetics.
We chose, investigated, and compared 7 WGA methods to evaluate their ability to amplify degraded and nondegraded DNA samples. They included PCR-based techniques: primer extension preamplification (PEP) PCR,4 degenerate oligonucleotide primed (DOP) PCR,5 GenomePlex WGA commercial kit (Sigma, St. Louis, Mo), and isothermal reaction–based methods, which utilize highly precise Phi29 polymerase with strand displacement activity: multiple displacement amplification (MDA),6 its commercial version (GenomiPhi Amplification Kit [Amersham Biosciences, Piscataway, NJ]), restriction and circularization aided rolling circle amplification (RCA-RCA),7 and blunt-end ligation–mediated (BL) WGA—the MDA modification utilizing T4 ligase and T4 polymerase.8
To our best knowledge, this is the first article comparing and analyzing in detail 7 different WGA methods as degraded and nondegraded DNA preamplification tool before Y-chromosome STR loci analysis.
MATERIALS AND METHODS
Human muscle tissue samples collected from a male thigh during autopsy (24 hours after death).
Biological DNA Degradation, Estimation of the Degradation Rate, and DNA Isolation
Tissue sample was degraded to the level of 200 base pairs (bp) and 100 bp. The degradation, the control of the degradation level, and DNA isolation were performed as described previously.11
DNA 100, 10, and 1 ng originating from degraded and nondegraded muscle tissues were subjected to amplification using 7 WGA methods: PEP-PCR,4 DOP-PCR,5 GenomePlex WGA (Sigma), MDA,6 GenomiPhi DNA Amplification Kit (Amersham Biosciences), RCA-RCA,7 and BL-WGA,8 with some modifications as described previously.11
Quantification of DNA
Quantification of DNA before and after WGA reaction was carried out as described previously.11
Polymorphic Loci Amplification and Evaluation of Electrophorograms
AmpFlSTR Yfiler (Life Technologies) for Y chromosome polymorphic loci was used as recommended by the manufacturer. 1 ng of DNA was added to the reaction. Amplification products were run on an ABI PRISM 3130 genetic analyzer. For each reaction, 1 μl of PCR product was added to 9 μl of master mix (8.7 μl Hi-Di formamide and 0.3 μl GeneScan-500 LIZ Size Standard, Life Technologies). Samples were processed using a FragmentAnalysis36_POP4 run module (POP-4 polymer, 5-second injection, 1.2-kV injection voltage, 60°C, runtime 1300 seconds, dye set G5). The results were analyzed using GeneMapper v.3.2 (Life Technologies). The analysis threshold value was established experimentally at the level of 50 relative fluorescence units.
RESULTS AND DISCUSSION
Muscle tissue was subjected to biological in vitro degradation until the DNA was degraded to approximately 200 and 100 bp, which mimics DNA degraded by environmental factors. Recently, we have described in detail comparison of the yields of amplification of WGA methods for both nondegraded and degraded DNA and possibility of obtaining SGMPlus profiles of autosomal DNA.11 Briefly, the highest increase in DNA amount was observed for RCA-RCA method for both degraded and nondegraded DNA, reaching even 12,000-fold increase for 1-ng input of nondegraded DNA. Similar increase was observed for BL-WGA. The lowest amplification efficiency was observed for PEP method. In general, we observed similar but insignificantly lower increase for degraded DNA than for nondegraded DNA.11
To evaluate the quality of WGA-preamplified DNA in terms of Y-chromosome STR analysis, samples of native DNA, degraded DNA (200 bp), and WGA-preamplified DNA were tested using Yfiler kit. The obtained profiles were subjected to detailed evaluation, especially in terms of accordance with native profiles, locus dropouts, and artifacts. Table 1 presents the impact of the amount of preamplified DNA on quality of Yfiler profiles when using different WGA methods.
Analysis of Yfiler Profiles of Nondegraded DNA Preamplified Using WGA
The best results for nondegraded DNA were obtained with PEP and GenomiPhi methods. They allowed recovery of complete profiles for all DNA concentrations (100, 10, and 1 ng of DNA input at WGA reaction) with no formation of additional peaks or stutters exceeding 20% of the relevant allele height. The MDA method allowed recovery of all alleles; however, additional peaks were observed for large quantities of DNA (100 and 10 ng). The DOP method gave complete profiles without artifacts, except for the highest concentration of DNA (100 ng), which resulted at recovery of 94% of the expected alleles; however, it could be due to the reaction overloading (Table 1).
In the remaining cases: GenomePlex, RCA-RCA, and BL-WGA, incomplete profiles were observed. GenomePlex allowed amplification of all loci for 100 and 10 ng of DNA input and 70.5% of the profile for 1 ng and produced additional peaks and numerous stutters, which reached 45% of the height of the correct allele. Stutter values were as follows: 34% (locus DYS 390, 100 ng), 43% (locus DYS 392, 10 ng), 45%, and 32% (locus DYS 391 and DYS 390, respectively, 1 ng). Furthermore, n-2, n-4, and n-6 stutters were formed (locus DYS 19, 10 ng), which in the case of n-2 fraction was up to 30%. Additional fractions n-4 (45% of the relevant peak height) and n-8 were observed also at the locus DYS 391 (1 ng) (Table 1; Fig. 1).
Both RCA-RCA and BL-WGA methods allowed the recovery of partial profiles (88.2%, 58.8%, 41% and 64.7%, 47.6%, 17.6% of full profile allelic coverage for 100, 10, 1 ng, respectively), without undesired peaks (Table 1).
Analysis of DNA profiles, obtained using different WGA methods, showed strong imbalance of the profiles and variations of levels of amplification of particular loci. Depending on the DNA input and the method used, different loci amplified with different strength. The ratios of height of the most and least amplified alleles were from 2-fold for BL-WGA to even 82- and 107-fold in the cases of GenomePlex and DOP (Table 2).
These results are mostly in accordance with those previously obtained for autosomal SGMPlus loci, where full recovery of alleles was observed for PEP, GenomiPhi, and DOP for all DNA concentrations. In the case of MDA, for SGMPlus, 95.2%, 90.4%, 95.2% of alleles (100, 10, and 1 ng of input DNA, respectively) were obtained, contrary to full profile recovery in the case of Yfiler. Alleles’ recovery observed for SGMPlus was as follows: for GenomePlex: 80.9%, 71.4%, 80.9%; for RCA-RCA: 95.2%, 76.6%, 14.2%; and for BL-WGA 85.7%, 38%, 4.7% (100, 10, and 1 ng, respectively), which indicate partial profiles like in the case of Yfiler. For SGMPlus, additional peaks were observed for GenomePlex, but also for RCA-RCA and BL-WGA. Elevated stutters, as in the case of Yfiler, were observed only for GenomePlex (up to 26%, 37%, and 48% for 100, 10, and 1 ng, respectively); n-4 and n-8 fractions were also present.11
These results show that some WGA methods can contribute to deterioration of DNA profile. Even GenomiPhi and DOP, which in our hands gave complete, expected profiles, according to Hanson and Ballantyne,3 do not improve the sensitivity of the analysis of autosomal and Y-chromosome STR loci compared with analysis without WGA.
Summing up, among the 7 analyzed WGA methods, the most satisfactory and reliable results for nondegraded DNA were obtained with PEP and GenomiPhi; however, the latter caused about 10 times higher increase in DNA amount.11 For both methods, 100% alleles’ recovery and no artifacts were observed for all analyzed DNA concentrations.
Analysis of Yfiler Profiles of Degraded DNA Preamplified Using WGA
In the case of degraded DNA (200 bp), alleles’ recovery, as expected, was remarkably reduced. Y-STRs are homozygous in nature and provide haplotype with 1 allele in most loci. This eliminates problems of intralocus peak height imbalance, which are common in the case of autosomal loci of DaLT DNA analysis.
No WGA method allowed recovery of complete Yfiler profile for DNA degraded to 200 bp (Table 1). However, the fullest amplification profile was observed for GenomePlex and PEP. In the case of GenomePlex, 70.5%, 52.9%, and 29.4% of expected alleles were amplified (100, 10, and 1 ng, respectively), and profiles revealed additional peaks and stutters reaching 37% and 26% (100 and 10 ng, respectively) (Fig. 2). Besides, quite high differences in the amplification strength of particular loci and preferential amplification of short alleles were observed (Table 2). For PEP method, no artifacts were obtained, but alleles’ recovery was lower: 70.5%, 29.4%, and 5.8% (100, 10, and 1 ng, respectively). Restriction and circularization aided rolling circle amplification allowed allele recovery from 58.8% to 5.8%, and BL-WGA from 35.2% to 0% with no artifacts. The remaining methods (DOP, MDA, GenomiPhi) did not produce amplification signals for degraded DNA (Table 1).
Similarly to nondegraded DNA, the results of Yfiler analysis of degraded DNA are analogous to previously obtained SGMPlus effects. In both cases, not even partial profile was obtained for DOP, MDA, and GenomiPhi. In the case of SGMPlus, for 100, 10, and 1 ng, respectively, the allelic coverage was as follows: for PEP method: 90.4%, 14.2%, and 4.7%; for GenomePlex: 90.4%, 23.8%, and 19%; for RCA-RCA: 76.2%, 9.5%, and 0%; and for BL-WGA: 42.8%, 52.3%, and 0%. Like in the case of Yfiler, for GenomePlex followed by SGMPlus typing, elevated stutters were observed but only for 100 ng. Moreover, for RCA-RCA and BL-WGA, additional peaks for 100 ng were present, which did not occur in case of Yfiler. In case of DNA degraded to 100 bp, analysis of Yfiler loci did not produce results, like in SGMPlus case.
Significant difference between effectiveness of degraded and nondegraded DNA analysis with GenomiPhi and MDA most probably results from the fact that both methods use hyperbranched amplification, which does not replicate fragments shorter than 1000 bp.7,8 Restriction and circularization aided rolling circle amplification and BL-WGA, which are based on rolling circle amplification, were developed as an alternative to or improvement of MDA techniques (GenomiPhi), allowing analysis of formalin-fixed paraffin-embedded samples7 and plasma-circulating DNA (apoptotic <200 bp and necrotic >5000 bp DNA)8 and, as our results show, may work also for DaLT DNA.
The influence of WGA methods on the quality of Y-STR profiling was investigated by Hanson and Ballantyne,3 who, using modified PEP method (mI-PEP), obtained DNA profiles from 3-month-old and 1-year-old environmentally impacted bloodstains. It is tough; however, to estimate the level of degradation at these stains and compare it with our results, because we had subjected biological material to degradation until it reached the length of 200 bp.
Analysis of Y chromosome is especially important at investigation of sexual offenses, but also in kinship testing (paternal linage) and clinical and historical research. Analysis approach of highly degraded, poor-quality, and poor-quantity DNA is one of the issues that must be improved. Among WGA methods analyzed here, GenomePlex and PEP produced the best results for degraded DNA. Methods based on rolling circle amplification can also give passable results at analysis of degraded DNA. In case of nondegraded DNA, the best results were obtained for GenomiPhi and PEP. These results are in accordance with those previously obtained for SGMPlus.
Comparison of the results of degraded and nondegraded DNA analysis using different methods allows application of the appropriate research approach, depending on the quality of analyzed material. Employment of a proper WGA technique can be very important at investigation and contribute to the detection of key evidence.
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