Linked Life Data

12095269

Source:http://linkedlifedata.com/resource/pubmed/id/12095269

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2002-7-3
pubmed:abstractText
The idea of modifying DNA with bisulfite has paved the way for a variety of polymerase chain reaction (PCR) methods for accurately mapping 5-methylcytosine at specific genes. Bisulfite selectively deaminates cytosine to uracil under conditions where 5-methylcytosine remains unreacted. Following conventional PCR amplification of bisulfite-treated DNA, original cytosines appear as thymine while 5-methylcytosines appear as cytosine. Because the relative thermostability of a DNA duplex increases with increasing content of G:C base pairs, PCR products originating from DNA templates with different contents of 5-methylcytosine differ in melting temperature, i.e., the temperature required to convert the double helix into random coils. We describe two methods that resolve differentially methylated DNA sequences on the basis of differences in melting temperature. The first method integrates PCR amplification of bisulfite-treated DNA and subsequent melting analysis by using a thermal cycler coupled with a fluorometer. By including in the reaction a PCR-compatible, fluorescent dye that specifically binds to double-stranded DNA, the melting properties of the PCR product can be examined directly in the PCR tube by continuous fluorescence monitoring during a temperature transition. The second method relies on resolution of alleles with different 5-methylcytosine contents by analysis of PCR products in a polyacrylamide gel containing a gradient of chemical denaturants. Optimal resolution of differences in melting temperature is achieved by a special design of PCR primers. Both methods allow resolution of "heterogeneous" methylation, i.e., the situation where the content and distribution of 5-methylcytosine in a target gene differ between different molecules in the same sample.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
1046-2023
pubmed:author
pubmed:copyrightInfo
(c) 2002 Elsevier Science (USA).
pubmed:issnType
Print
pubmed:volume
27
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
121-7
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Profiling DNA methylation by melting analysis.
pubmed:affiliation
Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. perg@cancer.dk
pubmed:publicationType
Journal Article