CHARACTERIZATION OF MITOCHONDRIAL-DNA USING LOW-STRINGENCY SINGLE SPECIFIC PRIMER AMPLIFICATION ANALYZED BY LASER-INDUCED FLUORESCENCE - CAPILLARY ELECTROPHORESIS

Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single n ucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method tha t does not require additional biochemical treatments. The analysis of LSSP-PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D-loop region is described. The DNA from five individuals were amplified (first step) using sequence-s pecific primers to produce 1021 bp fragments containing the D-loop reg ion. Each fragment was isolated by electroelution using CE and UV dete ction, and subjected to a second amplification (second step) using a s ingle primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolve d and analyzed by CE with the intercalator TOTO-1 and laser-induced fl uorescence (LIF) detection. The LSSP-PCR profiles were unique for each individual, indicating that this technique may be applicable for fore nsic identity testing.