ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping

Publication: Research - peer-reviewJournal article – Annual report year: 2008

View graph of relations

A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.
Original languageEnglish
JournalScandinavian Journal of Clinical & Laboratory Investigation
Publication date2008
Volume68
Issue8
Pages720-730
ISSN0036-5513
DOIs
StatePublished
CitationsWeb of Science® Times Cited: 0

Keywords

  • DNA fingerprinting, TspRI, genotype, Amplified fragment length polymorphism analysis, polymerase chain reaction
Download as:
Download as PDF
Select render style:
APAAuthorCBEHarvardMLAStandardVancouverShortLong
PDF
Download as HTML
Select render style:
APAAuthorCBEHarvardMLAStandardVancouverShortLong
HTML
Download as Word
Select render style:
APAAuthorCBEHarvardMLAStandardVancouverShortLong
Word

ID: 3416835