The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit

Cooksley, Joseph David, The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit. Master of Science (Forensic Genetics), August 2003, 93pp., 3 tables, 14 figures, 1 appendix, references, 17 titles. Nucleotide sequences from coding regions in the mitochondrial DNA genome were evaluated for use in designing an oligonucleotide probe specific to humans that could be used with Quantiblot Human DNA Identification kit from Applied Biosystems. Two probes were designed and evaluated using the conditions, reagents, and protocols recommended in the kit instructions, as well as with less stringent conditions than those recommended in the kits instructions. Probe 1 bound to amplified mtDNA at low stringency conditions, with greater probe amount, with heat denaturation of the probe, and more HRP-SA enzyme conjugate added than recommended in the protocol. Probe 1 did not hybridize to mtDNA extracted from a buccal swab, bloodstain cards, or a whole blood sample. Probe 2 did not show any hybridization to mtDNA when evaluated.