Description of original award (Fiscal Year 2023, $413,504)
Hair is a common piece of evidence found at crime scenes, but it has limited forensic value with the currently used methods. DNA extracted from the hair has both mitochondrial and nuclear components. While analysis of mitochondrial DNA can provide information about the source, limitations to mitochondrial DNA testing frequently prevent the method from achieving individualization. Nuclear
DNA from hair has not thus far been useful to standard forensic analyses because of limited quantity and quality. Conventional DNA analysis is based on the amplification via polymerase chain reaction (PCR) of short tandem repeat (STR) regions. The DNA extracted from hair is highly degraded, and the fragments are too small to yield the commonly used STR amplicons used in forensic genotype profiling.
Profiling using single nucleotide polymorphisms (SNPs) and small haplotypes is possible with the degraded fragments with shed hair, but has not been widely developed because of the small quantity of DNA that arises from extraction of shed hair.
This project proposes to develop a hybridization capture method for target enrichment using massively parallel sequencing techniques to analyze nuclear DNA in hair shafts. DNA will be extracted from rootless hair shaft using hair extraction protocols developed to recover small fragments, and library preparation will be conducted using Takara Bio and KAPA preparation kits for Illumina. Libraries will be
enriched for fragments containing SNPs and microhaplotypes of interest through hybridization with a specially designed panel of probes and protocols using the capture hybridization kit myBaits®. The enriched DNA will then be templated and sequenced on the Illumina MySeq. The panel of loci targeted by the probes consists of 18 small MHs and 220 SNPs yielding SNPs, yielding a power of discrimination
over ten orders of magnitude greater than that of a conventional STR assay and will enable ancestry prediction.
The combination of target enrichment and MPS will overcome the problems previously encountered with poor quality and quantity of hair DNA, and introduce the element of added accuracy when genotyping with the DNA sequence as compared to STR analysis, which is based only on the size of amplicons. The ability to generate nuclear DNA profiles from hair shafts will extend to other evidentiary materials characterized by degraded DNA, and will open another technical approach to forensic investigations. CA/NCF
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