Optimization of coupled PCR amplification and cycle sequencing of cloned and genomic DNA.
We describe optimization of a coupled amplification and cycle sequencing (CAS) method for rapid characterization of cloned or genomic DNA. Our modification of this method, termed coupled PCR amplification and cycle sequencing (CPACS), utilizes commercially available reagents, does not require template purification and produces high-quality sequence ladders from nanogram quantities of complex genomic DNA. The reactions have been streamlined to permit automation. Finally, we show that the technique can be applied more efficiently in conjunction with the AutoTrans 350 Direct Transfer Electrophoresis System and 33P-labeled sequencing primers.
Reynolds
T R
TR
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth Univ. Richmond 23298.
Uliana
S R
SR
Floeter-Winter
L M
LM
Buck
G A
GA
D001322:Autoanalysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004247:DNA