RT-PCR of long prokaryotic operon transcripts without DNase treatment.
RT-PCR is a powerful technique used in the amplification and detection of rare mRNAs. However, one of the most serious drawbacks of this method is the amplification of false-positive products due to DNA contamination in the RNA samples. This pitfall is particularly hard to overcome when RNA from prokaryotic origin is used. We present here a modification of the EXACT RT-PCR method that was successfully employed in the amplification of the low abundant full-length polycistronic pst operon mRNA of Escherichia coli. No DNase treatment of the RNA template is required, but unlike the original EXACT RT-PCR, a hybrid primer that is not composed of oligo(dT) was used. A nonhomologous sequence was incorporated at the reverse transcription step into the 5' end of the first-strand cDNA by means of the hybrid primer. For the PCR, a gene-specific primer and a second primer identical to the nonhomologous portion of the hybrid primer were used. To avoid amplification of genomic DNA, the hybrid-primer molecules that were not incorporated into the first-strand cDNA were removed by RNase H treatment followed by ultrafiltration.
Aguena
Meire
M
Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, São Paulo, SP 05508-900, Brazil.
Spira
Beny
B
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D009876:Operon / Q000235:genetics*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics; D028044:Phosphate-Binding Proteins; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016914:Ribonuclease H / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; C489729:PstA protein, E coli; C489730:PstC protein, E coli; C078409:PstC protein, bacteria; C090133:PstS protein, E coli; D012329:RNA, Bacterial; D012333:RNA, Messenger; C078408:phosphate-specific transport protein A, bacteria; D003851:Deoxyribonucleases; D016914:Ribonuclease H; D000251:Adenosine Triphosphatases; C120060:phosphate import ATP-binding protein, Bacteria