Linked Life Data

10092070

Source:http://linkedlifedata.com/resource/pubmed/id/10092070

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1999-4-13
pubmed:abstractText
PCR amplification and product analysis for the detection of chromosomal translocations such as bcl-1/JH have traditionally been performed as a two-step process with separate amplification and product detection. PCR product detection has generally entailed gel electrophoresis, hybridization, or sequencing for confirmation of assay specificity. By using a microvolume fluorimeter integrated with a thermal cycler and the PCR compatible double-stranded DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and detected bcl-1/JH translocation products by using rapid cycle PCR and fluorescence melting curve analysis. We analyzed DNA from 25 cases of lymphoproliferative disorders comprising 12 previously documented bcl-1/JH-positive mantle cell lymphomas, and 13 reactive lymphadenopathies. The samples were coded and analyzed in a blind manner for the presence of bcl-1/JH translocations by fluorescence melting curve analysis. The results of fluorescence analysis were compared with those of conventional PCR and gel electrophoresis. All of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at about 86 degrees C by melting curve analysis. For easier visualization of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T). Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 40-fold. Our results indicate that nucleic acid amplification integrated with fluorescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of specific PCR product detection without electrophoresis or expensive fluorescently labeled probes makes this methodology attractive for studies in molecular pathology.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0023-6837
pubmed:author
pubmed:issnType
Print
pubmed:volume
79
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
337-45
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma.
pubmed:affiliation
Department of Pathology, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
pubmed:publicationType
Journal Article