Linked Life Data

10608248

Source:http://linkedlifedata.com/resource/pubmed/id/10608248

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2000-1-20
pubmed:abstractText
Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) has been gaining popularity as a preferred method to determine the clonality of T-cell populations in small or sparsely infiltrated specimens such as skin biopsies. T-cell receptor (TCR)-gamma gene rearrangements are amplified using nested consensus primers in two rounds of PCR and then are separated by DGGE. Sensitivity is better than with conventional Southern blot analysis but not fully defined. In addition to a discrete primary band resulting from a monoclonal TCR-gamma gene rearrangement, there are often weaker secondary bands of unknown origin. Our goals were to define the PCR/DGGE clonal detection threshold, determine the genesis of the multiband pattern, and optimize methods to minimize extraneous bands. Titration studies showed that the sensitivity of PCR/DGGE for detecting clonal T-cell DNA is affected by the polyclonal T-cell content of the background DNA. The detection threshold is 0.001% using keratinocyte DNA as diluent but only 1% with tonsil DNA. Analysis of monoclonal T-cell lines showed that multiple bands can be produced by a single TCR-gamma gene rearrangement. Mixing of inner and outer primer pair PCR products showed that this is an artifact resulting from different sized PCR products produced during the two rounds of nested PCR required for optimal specificity. Repeat DGGE of isolated bands ruled out variable mobility of partially melted PCR products. Reduction of first round PCR product used as second round target from 10 microl to 1 microl, or a decrease of first round primers from 40 pmole to 5 pmole, resulted in diminished secondary bands without compromising primary band intensity. These results show that: 1) PCR/DGGE has a realistic clonal detection threshold of 0.1% to 1%, 2) multiple bands are consistent with a monoclonal T-cell population, and 3) conditions can be optimized to minimize artifactual secondary bands.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0193-1091
pubmed:author
pubmed:issnType
Print
pubmed:volume
21
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
547-51
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis, and methodologic optimization.
pubmed:affiliation
Department of Dermatology, Skin Diseases Research Center at Case Western Reserve University, Cleveland, Ohio, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.