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Type: Journal article
Title: Non-specific, nested suppression PCR method for isolation of unknown flanking DNA
Author: Tamme, R.
Camp-Dotlic, E.
Kortschak, R.
Lardelli, M.
Citation: Biotechniques, 2000; 28(5):895-902
Publisher: Eaton Publishing Co
Issue Date: 2000
ISSN: 0736-6205
Organisation: Centre for the Molecular Genetics of Development
Abstract: 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.
Keywords: Animals; Zebrafish; Alkalies; Monophenol Monooxygenase; Taq Polymerase; DNA, Complementary; Oligonucleotide Probes; Sensitivity and Specificity; Cloning, Molecular; Polymerase Chain Reaction; Nucleic Acid Hybridization; Transcription, Genetic; Regulatory Sequences, Nucleic Acid; Gene Dosage
RMID: 0001000493
DOI: 10.2144/00285st02
Appears in Collections:Molecular and Biomedical Science publications
Centre for the Molecular Genetics of Development publications

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