An approach for global scanning of single nucleotide variations.
Efficient global scanning of single nucleotide variations in DNA sequences between related, complex DNA samples remains a challenge. In the present article we present an approach to this problem. We have used immobilized thymidine DNA glycosylases to capture and enrich DNA fragments containing internal mismatched base pairs and separate these fragments as a pool from perfectly base-paired fragments as another pool. Enrichments of up to several hundredfold were obtained with one cycle of treatment, and all of the four groups of single nucleotide mismatches were fully covered by combining use of two thymine DNA glycosylases generated here. We have used a heterohybrid-orientating strategy for selective amplification of duplexes with one strand derived from each of two input DNA samples, which can also be used for selective amplification of duplexes with both strands derived from one of two input samples when desired. By combining these methods, the single nucleotide variations either between two DNA pools or within one DNA pool can be obtained in one process. This approach has been applied to the total cDNA from a human cell line and has several potential applications in mapping genetic variations, particularly global scanning of cDNA single nucleotide variations or polymorphisms, and finally high-throughput mapping of complex genetic traits.
Pan
Xinghua
X
Molecular Staging, Inc., 300 George Street, Suite 701, New Haven, CT 06511, USA.
Weissman
Sherman M
SM
D020137:Base Pair Mismatch; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics; D004800:Enzymes, Immobilized; D005821:Genetic Techniques; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000235:genetics
D018076:DNA, Complementary; D004800:Enzymes, Immobilized; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)