This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nisson, P E Articles by Watkins, P C Search for Related Content PubMed PubMed Citation Articles by Nisson, P E Articles by Watkins, P C Social Bookmarking                   What's this?

Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.

Life Technologies, Inc. Gaithersburg, Maryland 20877.

Abstract

By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-independent cloning by using UDG to create complementary single-stranded sticky ends between vector and Alu-PCR products generated from cosmid clones containing DNA from human chromosome 21. Using a single primer, Alu-PCR amplifies the sequence between appropriately oriented, repetitive (Alu) sequences in human DNA that are no more than 2 to 3 kb apart. Nineteen Alu-PCR products were observed in four human chromosome 21 cosmids. Thirteen of these products were detected among 48 subclones picked at random after cloning of the Alu-PCR products using UDG. The size or abundance of an Alu-PCR product did not appear to affect significantly the efficiency of cloning. Eight of the subclones were tested and all hybridized to human chromosome 21 DNA. UDG cloning should prove to be a general PCR cloning method that allows one to rapidly subclone small fragments from human genomic DNA.

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati