Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.
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.
Nisson
P E
PE
Life Technologies, Inc. Gaithersburg, Maryland 20877.
Rashtchian
A
A
Watkins
P C
PC
D001483:Base Sequence; D002891:Chromosomes, Human, Pair 21; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids; D004247:DNA / Q000235:genetics; D045647:DNA Glycosylases; D003856:Deoxyuracil Nucleotides; D005069:Evaluation Studies as Topic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D051981:Uracil-DNA Glycosidase
D003856:Deoxyuracil Nucleotides; D012313:RNA; D004247:DNA; C007267:2'-deoxyuridylic acid; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase