An RT-PCR artifact in the characterization of bacterial operons

During the course of experiments involving RT-PCR to investigate the co-transcription of the pet and res operons of the bacterium Acidithiobacillus ferrooxidans we observed that the mRNA of certain gene pairs can be amplified by RT-PCR in the absence of added RT-primer. We provide experimental evide...

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Autores principales:	Guacucano,Maritza, Levican,Gloria, S. Holmes,David, Jedlicki,Eugenia
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2000
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582000000300005
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spelling	oai:scielo:S0717-345820000003000052003-08-18An RT-PCR artifact in the characterization of bacterial operonsGuacucano,MaritzaLevican,GloriaS. Holmes,DavidJedlicki,Eugenia During the course of experiments involving RT-PCR to investigate the co-transcription of the pet and res operons of the bacterium Acidithiobacillus ferrooxidans we observed that the mRNA of certain gene pairs can be amplified by RT-PCR in the absence of added RT-primer. We provide experimental evidence that eliminates, as explanations for the observed results, the possible global contamination of the RNA preparation by genomic DNA or by contaminating single stranded DNA or RNA fragments. We suggest that the results can be explained by self-priming of the RNA perhaps as a result of secondary structure configurations that provide a suitable 3-terminus to prime the reverse transcriptase. This type of artifact can be recognized by carrying out a control in which exogenously added RT-primers are left out of the reaction. Such a control is essential if RT-PCR is to be used to investigate and correctly identify operons. RT-PCR is also an important tool for the amplification of eukaryotic mRNAs and our observations may also be applicable to these systems.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.3 n.3 20002000-12-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582000000300005en
institution	Scielo Chile
collection	Scielo Chile
language	English
description	During the course of experiments involving RT-PCR to investigate the co-transcription of the pet and res operons of the bacterium Acidithiobacillus ferrooxidans we observed that the mRNA of certain gene pairs can be amplified by RT-PCR in the absence of added RT-primer. We provide experimental evidence that eliminates, as explanations for the observed results, the possible global contamination of the RNA preparation by genomic DNA or by contaminating single stranded DNA or RNA fragments. We suggest that the results can be explained by self-priming of the RNA perhaps as a result of secondary structure configurations that provide a suitable 3-terminus to prime the reverse transcriptase. This type of artifact can be recognized by carrying out a control in which exogenously added RT-primers are left out of the reaction. Such a control is essential if RT-PCR is to be used to investigate and correctly identify operons. RT-PCR is also an important tool for the amplification of eukaryotic mRNAs and our observations may also be applicable to these systems.
author	Guacucano,Maritza Levican,Gloria S. Holmes,David Jedlicki,Eugenia
spellingShingle	Guacucano,Maritza Levican,Gloria S. Holmes,David Jedlicki,Eugenia An RT-PCR artifact in the characterization of bacterial operons
author_facet	Guacucano,Maritza Levican,Gloria S. Holmes,David Jedlicki,Eugenia
author_sort	Guacucano,Maritza
title	An RT-PCR artifact in the characterization of bacterial operons
title_short	An RT-PCR artifact in the characterization of bacterial operons
title_full	An RT-PCR artifact in the characterization of bacterial operons
title_fullStr	An RT-PCR artifact in the characterization of bacterial operons
title_full_unstemmed	An RT-PCR artifact in the characterization of bacterial operons
title_sort	rt-pcr artifact in the characterization of bacterial operons
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2000
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582000000300005
work_keys_str_mv	AT guacucanomaritza anrtpcrartifactinthecharacterizationofbacterialoperons AT levicangloria anrtpcrartifactinthecharacterizationofbacterialoperons AT sholmesdavid anrtpcrartifactinthecharacterizationofbacterialoperons AT jedlickieugenia anrtpcrartifactinthecharacterizationofbacterialoperons AT guacucanomaritza rtpcrartifactinthecharacterizationofbacterialoperons AT levicangloria rtpcrartifactinthecharacterizationofbacterialoperons AT sholmesdavid rtpcrartifactinthecharacterizationofbacterialoperons AT jedlickieugenia rtpcrartifactinthecharacterizationofbacterialoperons
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An RT-PCR artifact in the characterization of bacterial operons

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