title: Genetic disease detection and DNA amplification using cloned thermostable ligase.
abstract: Polymerase chain reaction, using thermostable DNA polymerase, has revolutionized DNA diagnostics. Another thermostable enzyme, DNA ligase, is harnessed in the assay reported here that both amplifies DNA and discriminates a single-base substitution. This cloned enzyme specifically links two adjacent oligonucleotides when hybridized at 65 degrees C to a complementary target only when the nucleotides are perfectly base-paired at the junction. Oligonucleotide products are exponentially amplified by thermal cycling of the ligation reaction in the presence of a second set of adjacent oligonucleotides, complementary to the first set and the target. A single-base mismatch prevents ligation/amplification and is thus distinguished. This method was exploited to detect 200 target molecules as well as to discriminate between normal beta A- and sickle beta S- globin genotypes from 10-microliters blood samples.
authors.affiliation: Department of Microbiology, Cornell University Medical College, New York, NY 10021.
authors.forename: F
authors.initials: F
authors.lastname: Barany
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D013824:Thermus / Q000201:enzymology / Q000235:genetics
chemical_list: D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D005914:Globins; D011088:DNA Ligases