PCR regimen for enhanced specificity and yield of targeted genomic DNA sequences: ras and p53.
By weighting the PCR reaction in favor of specificity for the target sequence in the beginning cycles and for continued efficient amplification of the sequence into later cycles, we were able to show an improvement in the specificity and quantity of amplified ras and p53 sequences. Increased purity and yield of specific products favorably enhanced post-PCR evaluation and interpretation of results using direct sequencing and single-stranded conformation polymorphism (SSCP) analysis when point mutations were present in DNA from tumor cell lines and tissues.
Phillips
D J
DJ
Hematologic Diseases Branch, Centers for Disease Control, Atlanta, Georgia 30333.
Benson
J M
JM
Pruckler
J M
JM
Hooper
W C
WC
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004277:DNA, Single-Stranded / Q000235:genetics; D016158:Genes, p53; D011905:Genes, ras; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides