Automated DNA sequencing methods involving polymerase chain reaction.
Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.
McBride
L J
LJ
Applied Biosystems, Foster City, CA 94404.
Koepf
S M
SM
Gibbs
R A
RA
Salser
W
W
Mayrand
P E
PE
Hunkapiller
M W
MW
Kronick
M N
MN
D001322:Autoanalysis; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D013698:Templates, Genetic
D006683:HLA-DQ Antigens; C051568:HLA-DQ1 antigen; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase