Genetic disease detection and DNA amplification using cloned thermostable ligase.
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.
Barany
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Department of Microbiology, Cornell University Medical College, New York, NY 10021.
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
D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D005914:Globins; D011088:DNA Ligases