Another approach that may show promise for limited-quantity DNA samples is the use of whole-genome amplification (WGA) (32,33). Ideally, the WGA method amplifies all the DNA in a sample in an unbiased fashion, yielding substantially greater quantities of DNA that can be subsequently analyzed using standard forensic assays. WGA methods, however, are subject to some of the same stochastic effects that LCN typing encounters. One WGA technique known as rolling circle amplification (RCA) (34), which uses a circular DNA template, could possibly obviate some of the stochastic limitations. With a highly processive polymerase, RCA can yield microgram quantities of DNA from circular templates and—because of the phenomenon of strand displacement—produce many copies of the same target molecule. However, with the exception of intact mitochondrial DNA (mtDNA), circular DNA fragments do not occur naturally in humans, and DNA is often highly degraded in human remains. Therefore, to better exploit RCA, the fragmented DNA in an evidence sample could be circularized (35). One example of a potentially useful ligase is CircLigase (Epicentre Biotechnologies, Madison, WI, USA), which possesses a catalytic activity that circularizes single-stranded DNA. It offers the potential for RCA to be used on compromised DNA materials. In effect, highly fragmented DNA that was a poor substrate for PCR can be converted into circular DNA and hence become a good template for RCA.
Repairing the DNA of lesions that have occurred after exposure to environmental insults is potentially a viable approach for increasing the number of template molecules obtained from degraded DNA samples. Some DNA repair kits have begun to reach the scientific community, such as PreCR Repair Mix (New England Biolabs, Ipswich, MA, USA). WGA might then be possible by employing linear multiple strand displacement.Improvements to typing low-quality samples Mini-STRs
Forensic samples are often compromised to the point where obtaining DNA typing results is challenging or not possible. Highly degraded samples that contain DNA fragments that are too short in length cannot be used to generate amplicons that are longer in length. Whether for LCN (to increase the number of available template molecules) or for conventional DNA typing (where the sample is degraded), reducing the size of the amplicons generated during the PCR will increase typing success by providing more efficient amplification.
The STR loci are currently the most informative genetic markers for identity testing. To improve success in STR typing with degraded DNA, increase sensitivity of detection, and enhance robustness or the quality of the assay, the PCR primers for the STR loci can be repositioned so they reside closer to the repeat (i.e., polymorphic) region (36). Thus, the amplified PCR products will be reduced in length, and if smaller than some of the fragmented DNA template molecules, genetic characterization of the sample may then be possible (Figure 1). Mulero et al. (37) described the conversion of eight STRs (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA) into mini-STRs and the development of the commercial AmpFlSTR MiniFiler PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). The amplicon range for the mini-STRs spans 71–250 bp in length. Since the amplicon products of mini-STRs will overlap in size more so than those in conventional STR kits, the four fluorescent dye tag system was increased to five dyes. Thus, the mini-STRs could be accommodated into one multiplex analysis. It would make sense that all STR kits be reconfigured into mini-STR kits for routine analysis of forensic evidence. Only one attempt would be needed to obtain a DNA profile, if possible, for a degraded, limited-quantity sample. More efforts will continue to convert all current forensically relevant STRs into mini-STRs. To do so and to incorporate at least the core forensic STRs in one multiplex amplification kit, it is likely that fluorescent capillary electrophoresis–based systems will require additional tags or dyes to resolve more size-overlapping mini-STRs. Likely additional novel mini-STRs will be sought; some already have been recommended. For example, Gill et al. (38) described the acceptance of three new mini-STRs (D10S1248, D14S1434, D22S1045) into the European standard Interpol loci which now comprise 10 STR loci.