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Characterization of mitochondrial DNA using lowstringency single specific primer amplification analyzed by laser induced fluorescence – capillary electrophoresis
Author(s) -
Marino Michael A.,
Weaver Kristal R.,
Tully Lois A.,
Girard James E.,
Belgrader Phillip
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170915
Subject(s) - primer (cosmetics) , microbiology and biotechnology , capillary electrophoresis , primer dimer , polymerase chain reaction , biology , mitochondrial dna , dna , in silico pcr , genetics , multiplex polymerase chain reaction , chemistry , gene , organic chemistry
Polymerase chain reaction (PCR)‐based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide 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 that 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‐specific primers to produce 1021 bp fragments containing the D‐loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO‐1 and laser‐induced fluorescence (LIF) detection. The LSSP‐PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.