Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaTwo new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
[21] Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reactionKary B. Mullis, Fred Faloona|Methods in enzymology on CD-ROM/Methods in enzymology|1987 Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain ReactionKary B. Mullis, Fred Faloona, S. Scharf et al.|Cold Spring Harbor Symposia on Quantitative Biology|1986 The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods.
Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. 1985.Recent advances in recombinant DNA technology have made possible the molecular analysis and prenatal diagnosis of several human genetic diseases. Fetal DNA obtained by aminocentesis or chorionic villus sampling can be analyzed by restriction enzyme digestion, with subsequent electrophoresis, Southern transfer, and specific hybridization to cloned gene or oligonucleotide probes. With This disease results from homozygosity of the sickle-cell allele (rS) at the 3globin gene locus. The S allele differs from the wild-type allele (3A) by substitution of an A in the wild-type to a T at the second position of the sixth codon of the p chain gene, resulting in the replacement of a glutamic acid by a valine in the expressed protein. For the prenatal diagnosis of sickle cell anemia, DNA ob-
Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction