Generation of minisatellite variant repeat codes on an automated DNA sequencer using fluorescent dye-labeled primers.
We demonstrate the potential to generate unambiguous minisatellite variant repeat PCR profiles on an automated DNA sequencer using fluorescent dye-labeled primers. The products of a 24-cycle PCR amplification using 100 ng of template DNA were analyzed by conventional blot hybridization and also on an Applied Biosystems 373A DNA Sequencer. The results from the sequencer were clear to the 20th repeat when using a primer labeled with 6-FAM, whereas conventional analysis allowed codes to be defined to in excess of the 60th repeat. Use of a modified Tag primer sequence, which eliminates the potential for self-annealing, did not improve the clarity of the results under the PCR cycling conditions used.
Metropolitan Police Forensic Science Laboratory, London, UK.
B J
BJ
McKeown
G J
GJ
Lyndon
J F
JF
Andersen
D001322:Autoanalysis; D001483:Base Sequence; D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes