Open AccessInternal and Flanking Sequence from AFLP Fragments Using Ligation-Mediated Suppression PCR
Author(s) -
James M. Schupp,
Lance B. Price,
Alexandra M. Klevytska,
Paul Keim
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99265st04
Subject(s) - amplified fragment length polymorphism , biology , genetics , subcloning , polymerase chain reaction , sequence analysis , restriction fragment , cleaved amplified polymorphic sequence , restriction fragment length polymorphism , computational biology , microbiology and biotechnology , dna , gene , genetic diversity , plasmid , population , demography , sociology
Amplification fragment-length polymorphism (AFLP) analysis has proven to be a powerful tool for developing a large number of reliable genetic markers across a wide variety of organisms. Often it is desirable to further characterize these markers by obtaining internal and flanking sequence information. Here, we present a systematic approach for obtaining such information from AFLP markers. AFLP fragments can be isolated from dried polyacryamide sequencing gels (that have been stored for extended periods of time), amplified using PCR and subjected to sequence analysis. Outwardly oriented locus-specific primers are designed from the internal sequence and used in conjunction with adapter primers to amplify unknown regions that flank the internal sequence from up to 22 different restriction-ligation (R-L) reactions. This often results in multiple reactions yielding products of appropriate size and specificity for direct sequencing without the need for a nested PCR, extensive gel purification or subcloning. The detailed protocol is presented with PCR results from a variable AFLP fragment from Bacillus anthracis.