Nonspecific, nested suppression PCR method for isolation of unknown flanking DNA.
We report the development of a simple, sensitive and robust two-step PCR method for the isolation of unknown sequences flanking characterized regions of genomic DNA or cDNA. The method requires 100 bp or less of a known sequence upstream of an oligonucleotide primer binding site. A first round of suppression PCR is conducted at low stringency with a polymerase lacking exonuclease activity to generate a mixture of products including fragments of the desired flanking sequence that are often greater than 1 kb in length. The desired fragments are then amplified from the mixture in a second round of suppression PCR using an extended oligonucleotide in combination with a polymerase exhibiting exonuclease activity. These fragments are subsequently identified by hybridization with the 100 bp of known sequence or simply by cloning and sequencing. The method is widely applicable and allows isolation of novel cDNA from very low abundance transcripts.
Tamme
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R
Department of Genetics, University of Adelaide, Australia.
Camp
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E
Kortschak
R D
RD
Lardelli
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M
D000468:Alkalies; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification; D018628:Gene Dosage; D014442:Monophenol Monooxygenase / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014158:Transcription, Genetic / Q000235:genetics; D015027:Zebrafish
D000468:Alkalies; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D014442:Monophenol Monooxygenase; D019914:Taq Polymerase