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Automated amplification and sequencing of human mitochondrial DNA
Author(s) -
Sullivan Kevin M.,
Hopgood Romellé,
Lang Brian,
Gill Peter
Publication year - 1991
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.1150120105
Subject(s) - mitochondrial dna , primer (cosmetics) , hypervariable region , multiple displacement amplification , polymerase chain reaction , biology , dna , base pair , microbiology and biotechnology , dna sequencing , dna sequencer , genetics , inverse polymerase chain reaction , primer dimer , in silico pcr , sequence (biology) , computational biology , nested polymerase chain reaction , multiplex polymerase chain reaction , chemistry , gene , dna extraction , organic chemistry
Part of the human mitochondrial D‐loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M 13–21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single‐stranded DNA was generated. This was then sequenced directly by the dideoxy chain termination method using dye‐labelled universal sequencing primers in conjunction with a fluorescence‐based DNA sequencer. This enabled a 403‐base‐pair hypervariable segment of the D‐loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.