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Media type:
E-Article
Title:
Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences
Contributor:
Scharf, Stephen J.;
Horn, Glenn T.;
Erlich, Henry A.
Published:
American Association for the Advancement of Science (AAAS), 1986
Published in:
Science, 233 (1986) 4768, Seite 1076-1078
Language:
English
DOI:
10.1126/science.3461561
ISSN:
1095-9203;
0036-8075
Origination:
Footnote:
Description:
A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human β-globin gene and a 242-base pair fragment of the human leukocyte antigen DQα locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5′ ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.