Linked Life Data

15727129

Source:http://linkedlifedata.com/resource/pubmed/id/15727129

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2005-2-24
pubmed:abstractText
A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomonas citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIB17 hybrid primer, which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN <KAN-2> transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0736-6205
pubmed:author
pubmed:issnType
Print
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
225-9
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers.
pubmed:affiliation
Universidade de São Paulo, São Paulo, SP, Brazil.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Evaluation Studies