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Science 5 September 1986:
Vol. 233. no. 4768, pp. 1076 - 1078
DOI: 10.1126/science.3461561
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Articles

Science, Vol 233, Issue 4768, 1076-1078
Copyright © 1986 by American Association for the Advancement of Science

articles

Direct cloning and sequence analysis of enzymatically amplified genomic sequences

SJ Scharf, GT Horn, and HA Erlich

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 beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha 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.


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