In this work, we wished to investigate whether real-time TaqMan assays validated for use with low copy number (1 ng, 0.1 ng and 0.01 ng) or severely degraded genomic DNA might be of value for optimizing the performance of genotyping of MDA DNA.
To address this issue, we tested two independent protocols using our recently-developed forensic panel of SNPs. The first protocol revealed dissimilar results when genomic and MDA DNA were analyzed. In particular, 14 and 30 genomic DNA samples were not amplified with 0.1 ng and 0.01 ng dilutions, respectively, but all genotypes were concordant with the controls. In contrast, only one, one and 20 MDA DNA samples failed to be amplified with the 1 ng, 0.1 ng and 0.01 ng input dilutions, respectively. However, one MDA DNA sample (for inputs of 1 ng and 0.1 ng) revealed a genotype discordant with the controls, and 36 MDA DNA samples (for input of 0.01 ng) gave results dissimilar to the sequenced controls.
All discordant genotypes were false homozygous samples resulting from the complete failure of amplification of one allele in MDA (allele dropout, ADO) [7
]. We therefore decided to use a different master mix specifically designed to optimize the preferential binding of the allele-specific probe. We repeated the experiments and obtained better results. We observed 100% concordance and call rate for all dilutions of genomic DNA and for inputs of 1 ng and 0.1 ng in MDA. Furthermore, when inputs of 0.01 ng were analyzed, we observed a single PCR failure and only two samples gave results dissimilar to the controls. The resulting concordance and call rate were therefore increased to 99.923% for a 0.01 ng input in the MDA reaction, resulting in a probability of genotyping error of 7.7 × 10-4
, quite similar to that reported for STRs. Such results suggest that in combination with extra-short TaqMan assays and the specifically-designed master mix, MDA gave optimal findings in terms of sensitivity, reproducibility and robustness of genotyping. Independent studies have suggested that replicate MDA reactions should be pooled prior to genotyping in order to overcome the frequency of ADO and/or genotyping errors [25
]. Tzvetkov observed that the concordance between MDA and gDNA genotypes shows a small but significant improvement (0.5%) in pooled samples compared to single MDA. In our opinion, this strategy could be applied when sufficient DNA template is available.
Nevertheless, the comparison between genomic and amplified DNA showed that MDA does not improve our ability to type LCN DNA for single SNPs, as demonstrated by the better concordance and PCR failure rates in genomic than in MDA DNA. The genotype concordance and PCR failure rates were 100% and 99.923% in MDA DNA and genomic DNA respectively. However, it should be noted that MDA amplification produces large amounts of DNA, so it is possible to type many SNPs; in contrast, genomic DNA is often present in limited quantities so fewer SNPs can be typed. Finally, MDA allowed us to type a large number of SNPs starting from LCN DNA as template. Moreover, our experiments on artificially-degraded DNA provide evidence that MDA can enhance our capacity to type severely-degraded samples. In our experiments, nine samples failed to be amplified in genomic DNA but were successfully typed in the amplified DNA. We observed 100% concordance and call rate for all SNPs using MDA on degraded DNA. The quality of our results in typing digested DNA by MDA was surprising, although this has also been reported to a lesser extent for STR typing [16
]. Even though we typed completely-digested DNA (the average length of the degraded DNA (180s) was 80 bp down to less than 50 bp), it is likely that a small number of remaining intact fragments of template were amplified by MDA. In our opinion, these results depend on the marker types, the amplicon sizes and the genotyping technique. The results confirm and extend previous comparative analyses of SNP and STR typing in degraded forensic DNA (without MDA), demonstrating that good markers for degraded DNA depend on a small amplicon size [26
]. Here, very small amplicons (77 bp on average) have been typed by the most sensitive genotyping assay.
Finally, we recommend using MDA in order to overcome limitations of DNA quantity (when the amount of DNA is limited) or when only poor-quality DNA samples are available (severely-degraded samples, to increase the number of typed loci).
We would also point out that 0.26 ng of genomic DNA (0.01 ng for each locus) is needed to type all 21 autosomal SNPs of our panel and 5 extra SNPs [5
] successfully. It is also noteworthy that under these conditions, even though Real-Time PCR cannot allow multiplexing of the PCR, the amount of DNA needed to type a panel of SNPs in singleplex is comparable to current forensic STR methods and is much less than is needed to type forensic SNPs, as reported elsewhere [27
]. As a result, its use in forensic practice should be seriously considered when traditional forensic kits fail.
If the amount of DNA proves insufficient to type all SNPs independently, it will be possible to apply MDA even in LCN templates. In this case, only 0.01 ng of input DNA is needed to type a large number of SNPs (even more than the 26 reported here). This amount of DNA is much less than needed in the forensic protocols reported to date, so it represents a significant improvement in current forensic DNA protocols. As mentioned above, the possibility of a technical artefact (i.e. ADO) or a mis-genotyping following MDA with 0.01 ng of input DNA is 7.7 × 10-4. This is a lower error rate than that reported for the STRs used for forensic typing. Moreover, the mutation rate is higher in STRs than in SNPs: germline mutations at these STR loci lead to problems in the interpretation of genetic profile results.
In order to compare SNP typing between two or more samples, we can state – by analogy with STR analysis – that there is a match (biological compatibility) when there are no differences at genotypic level for any of the SNPs tested. In contrast, if SNP genotypes show differences, we can state that the samples considered originated from different sources (exclusion). Finally, if SNP genotypes show just a few differences (one or two), we can state that the data are inconclusive because the information is ambiguous or insufficient to support any conclusion, since genotyping errors or ADO events cannot be excluded. Our results suggest that the probability of two genotyping errors or ADOs in the same sample is 5.9 × 10-7 when only 10 pg of DNA are analyzed. Although this probability is quite low, homozygous genotypes in LCN DNA should be used with caution to exclude a match (compatibility): in this case, additional heterozygous markers should also be considered.