Molecular Tools for Low-Template DNA Analysis

DNA profiling by short tandem repeat (STR) analysis is a powerful tool for obtaining genetic information from biological material, to aid in the unambiguous identification of individuals to link persons, places, and objects. While the standard STR analysis methods work very well for relatively abundant and fresh or well preserved samples, it is often difficult to obtain complete genetic profiles from low-template samples (also referred to as low-copy-number samples) that may only have a few cells' worth of genetic material. In addition to the complete loss of genetic loci and the dropout of individual alleles, the profiles obtained from low-template samples suffer from increased stutter peaks, allelic imbalance, and allele drop-in resulting from stochastic effects induced during the polymerase chain reaction (PCR). Analysis is especially difficult for low-template samples that have been exposed to environmental factors that affect DNA integrity. GE Global Research is proposing to develop improved tools for examining such limited and/or damaged genetic evidence. Specifically, we propose to adapt two separate, highly sensitive methods for the treatment of low-template samples that would pre-amplify DNA from a few copies to an amount sufficient for quantification and robust profiling in addition to archiving. One method improves upon whole-genome amplification, using the technique of multiple displacement synthesis by Phi29 polymerase. This polymerase has excellent processivity and strand-displacement properties that enable high-fidelity amplification using random hexamer primers. We have made several improvements to the commercially available Genomi- PhiTM DNA amplification kit that include 1) self-cleaning of the reagents to eliminate potential contaminating DNA and 2) optimization of the formulation and hexamer composition to enable rapid, highly sensitive amplification of trace input DNA. The second method is a novel locus-specific isothermal reaction that involves incubation with nucleotide analog-modified primers, the genetically engineered nicking enzyme Endonuclease V, and a strand-displacing polymerase. An advantage of this isothermal method is that the amplification takes place at a lower temperature than conventional PCR, and so the degree of STR stutter peak formation is expected to decrease because of less DNA strand slippage. In addition, because amplification products are not templates for further amplification, the kinetics are linear, which is potentially advantageous for the maintenance of allelic ratios. This method could be adapted to pre-amplify only the desired STR loci from low-template DNA samples, providing a substantially increased probability of extracting a more complete profile. Combined with previously developed integrated methods to repair some of the DNA template lesions and fragmentation (NIJ 2006- DN-BX-K018), which are caused by environmental exposure and sample processing, we expect that this will greatly improve the ability to obtain complete genetic profiles from low template samples to aid in the investigation of felony crimes or for identification and chain-of- custody purposes. The work will be directed toward producing optimized protocols and recommendations for sample-specific use of the technologies. The methods will then be validated using standard forensic workflows and compared with current low-template methodology. ca/ncf