Long RT-PCR Amplification of the entire coding sequence of the polycystic kidney disease 1 (PKD1) gene.
Characterization of mutations of the PKD1 gene has been limited by the fact that three-fourths of this gene at its 5' end is homologous to sequences of at least three other genes on the same chromosome. We have therefore developed a method of long reverse transcription PCR for selective amplification of the entire coding sequence of the PKD1 gene from its mRNA. A PCR primer specific to the sequence in the 3' unique region of the PKD1 gene was synthesized for use coupled with a primer binding to sequence in the homologous region at a distance of about 13.6 kb apart. The commercial availability of RNase H-free reverse transcriptase for long cDNA synthesis and of an enzyme mixture containing Taq and Pfu DNA polymerases for long-range PCR have made this development possible. The long PCR product was proven to be derived from PKD1-mRNA. The results clearly indicated that the long PCR product contained the coding sequence derived from PKD1-mRNA. To our knowledge, this is the first report of a procedure that can reproducibly isolate full-length PKD1 coding sequence from its mRNA transcript, which will prove useful for screening and characterization of mutations in the PKD1 gene.
Thongnoppakhun
W
W
Faculty of Medicine-Siriraj Hospital, Bangkok, Thailand.
Wilairat
P
P
Vareesangthip
K
K
Yenchitsomanus
P T
PT
D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050396:TRPP Cation Channels
D017931:DNA Primers; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary; D011506:Proteins; D012333:RNA, Messenger; D050396:TRPP Cation Channels