RT-PCR of long prokaryotic operon transcripts without DNase treatment

J Microbiol Methods. 2003 Nov;55(2):419-23. doi: 10.1016/s0167-7012(03)00192-1.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / genetics
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Deoxyribonucleases / chemistry
  • Deoxyribonucleases / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Operon / genetics*
  • Periplasmic Binding Proteins / chemistry
  • Periplasmic Binding Proteins / genetics
  • Phosphate-Binding Proteins
  • RNA, Bacterial / genetics*
  • RNA, Bacterial / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Ribonuclease H / metabolism

Substances

  • ATP-Binding Cassette Transporters
  • Bacterial Proteins
  • Escherichia coli Proteins
  • Periplasmic Binding Proteins
  • Phosphate-Binding Proteins
  • PstA protein, E coli
  • PstC protein, E coli
  • PstC protein, bacteria
  • PstS protein, E coli
  • RNA, Bacterial
  • RNA, Messenger
  • phosphate-specific transport protein A, bacteria
  • Deoxyribonucleases
  • Ribonuclease H
  • Adenosine Triphosphatases
  • phosphate import ATP-binding protein, Bacteria