A method for selective PCR-amplification of genomic DNA fragments (SAGF method)
- Institute of Theoretical and Experimental Biophysics, Pushchino (Russian Federation)
- Branch of Shemyakin and Ovchinnikov Institute of Bioorganic, Pushchino (Russian Federation)
- Institute of Protein Research, Pushchino (Russian Federation)
A method is suggested for dividing into individual sets of the complex mixtures of fragments obtained by DNA cleavage with type IIS and IIN restriction endonucleases producing single-stranded termini with different sequences at the DNA fragment ends. The method is based on the ligation of short double-stranded adapters with single-stranded ends complementary to termini of the selected set of fragments followed by PCR-amplification with the primer representing one of the adapter chains. Using endonucleases BcoKI and Bli736I, recognizing sequences CTCTTC and GGTCTC and producing three- and four nucleotide 5{prime}-termini, respectively, it has been shown that amplification of a set of fragments occurs only upon attachment of the adapters to the DNA fragments with DNA-ligase. Several possible applications of the SAGF method are suggested: obtaining individual bands in DNA fingerprinting; reducing the kinetic complexity of DNA in representative difference analysis (RDA method) of complex genomes; cataloging of DNA fragments; construction of physical genome maps. 13 refs., 3 figs., 2 tabs.
- OSTI ID:
- 161890
- Journal Information:
- Biochemistry (New York), Journal Name: Biochemistry (New York) Journal Issue: 9 Vol. 60; ISSN 0006-2979; ISSN BIORAK
- Country of Publication:
- United States
- Language:
- English
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