Linked Life Data

2113558

Source:http://linkedlifedata.com/resource/pubmed/id/2113558

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1990-8-1
pubmed:abstractText
The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0022-1759
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
130
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
49-55
pubmed:dateRevised
2005-11-17
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction.
pubmed:affiliation
Department of Molecular Biology, Ciba-Geigy AG, Basel, Switzerland.
pubmed:publicationType
Journal Article