Construction of an internal standard for semiquantitative polymerase chain reaction analysis of heat shock proteins.
We describe a simple and effective method for generating competitive cDNA fragments for use as an internal standard in semi-quantitative polymerase chain reaction (PCR). The use of a nested composite primer to a region slightly within the expected amplicon but with heat shock protein 60 kDa (hsp60) primer sequence appended to the 5' terminus of the primer produces a "mimic" DNA that will model the PCR kinetics of the target template in sequence context, and PCR primer site, and that will be similar in size. This ensures that both competitor and target template are subjected to similar PCR kinetics and so allows more meaningful quantitation.
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Middlesex, UK.
M A
MA
Khan
N
N
Latif
P
P
Petrobelli
M H
MH
Yacoub
M J
MJ
Dunn
D001483:Base Sequence; D001706:Biopsy; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000473:pathology; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D006360:Heat-Shock Proteins