Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer | Zendy

Mogens Kilstrup | Zendy

Open Access

Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer

Author(s) -

Mogens Kilstrup

Publication year - 2000

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/28.11.e55

Subject(s) - biology , primer (cosmetics) , polymerase chain reaction , genome , microbiology and biotechnology , primer extension , dna , primer dimer , multiple displacement amplification , inverse polymerase chain reaction , multiplex polymerase chain reaction , genetics , gene , base sequence , dna extraction , chemistry , organic chemistry

In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer. The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments. After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5'-end only. Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used. As an example we report the amplification of the era gene from Lactococcus lactis.

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