Linked Life Data

15937116

Source:http://linkedlifedata.com/resource/pubmed/id/15937116

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
24
pubmed:dateCreated
2005-6-15
pubmed:abstractText
Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-10734205, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-11751232, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-11846375, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-11962616, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-12142390, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-14614044, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-14769930, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-1718662, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-3174659, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-8510538, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-8639506, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-9056205, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-9265640, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-9439442, http://linkedlifedata.com/resource/pubmed/commentcorrection/15937116-9465037
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0027-8424
pubmed:author
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
102
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8609-14
pubmed:dateRevised
2009-11-18
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.
pubmed:affiliation
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA. pierce@brandeis.edu
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't