New primer strategy improves precision of differential display.
To increase the reproducibility and to reduce the false positives in the initial mRNA differential display, modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA differential display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA differential display while still keeping the characteristics of the original method.
Zhao
S
S
Dana-Farber Cancer Institute, Boston, MA, USA.
Ooi
S L
SL
Pardee
A B
AB
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger