Direct sequencing of enzymatically amplified human genomic DNA.
The polymerase chain reaction is a recently described technique that uses flanking oligonucleotide primers and repeated cycles of enzymatic primer extension to amplify a short segment of DNA by greater than 100,000-fold. By use of sequencing primers located internal to the amplification primers, direct genomic sequence was obtained from enzymatically amplified DNA by using the dideoxynucleotide chain-termination method. The method is relatively simple and offers significant advantages in identifying mutations in genes for which the normal sequence is known. Heterozygous and homozygous mutations in the human beta- and gamma-globin loci were unambiguously identified in 3 days with less than 1 microgram of genomic DNA.
Engelke
D R
DR
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
Hoener
P A
PA
Collins
F S
FS
D004247:DNA / Q000235:genetics*; D005784:Gene Amplification; D005796:Genes; D005914:Globins / Q000235:genetics*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic
D005914:Globins; D004247:DNA