Quantitation and characterization of human nuclear and mitochondrial DNA with PCR and capillary electrophoresis

Scope and Method of Study: Mitochondrial DNA (mtDNA) sequencing is a valuable tool for identification in the forensic laboratory. Quantifying and characterizing the DNA in a sample, whether nuclear or mitochondrial, can assist an analyst in deciding which DNA typing method to use to yield probative results. One method developed here for quantifying and characterizing nuclear DNA known as Qualitative Template Amplification Technology, or Q-TAT, was recent shown to quantify and characterize nDNA recovered from evidentiary samples (Wilson et.al., J. For. Sci 55: 1050-1057). The goal of this research was to enhance the capability of the Q-TAT assay to include primers targeting mtDNA sequences that will allow for the quantitation and characterization of both nuclear and mitochondrial DNA in a single assay. Primers targeting two regions of the mtDNA close to the origin of replication were identified that direct the amplification of 97 basepair and 287 basepair sequences (mt97 and mt287 respectively).;Findings and Conclusions: Fluorescence incorporated into the mt97 and mt287amplicons reflected the amount of mtDNA recovered from a sample and the ratio of fluorescence incorporated into the 287 basepair versus the 97 basepair products reflected the extent of degradation of the mtDNA recovered. Standard plots of fluorescence in the 97 basepair or 287 basepair products versus known amounts of amplified human DNA showed good linearity from 15 pg to 1000 pg (R2 = >0.95), and the 287 basepair/97 basepair ratio of fluorescence decreased as the mtDNA template was increasingly degraded. The Q-TAT assay has also been used to quantify and characterize DNA in blood, hair, semen, bone and buccal swabs. The modified Q-TAT assay holds great promise as an effective tool for the DNA Analyst in evaluating forensic DNA evidence in deciding which DNA typing method approach is most likely to produce probative test results.