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Related Experiment Videos

Evidence for chimeric sequences formed during random arbitrarily primed PCR.

In Seop Chang1, Jimmy D Ballard, Lee R Krumholz

  • 1Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019-0245, USA.

Journal of Microbiological Methods
|July 5, 2003
PubMed
Summary
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Chimeric sequences frequently appeared in random arbitrarily primed polymerase chain reaction (RAP-PCR) experiments. This finding is crucial for accurately interpreting RAP-PCR data in gene expression studies.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genomics

Background:

  • Sulfate-reducing bacteria (SRB) are crucial in biogeochemical cycles.
  • Random arbitrarily primed polymerase chain reaction (RAP-PCR) is a common technique for gene expression analysis.
  • Understanding gene expression in SRB is vital for environmental and industrial applications.

Purpose of the Study:

  • To investigate the occurrence and nature of chimeric sequences in RAP-PCR experiments.
  • To determine the origin of these chimeric sequences in the context of SRB gene expression analysis.

Main Methods:

  • Utilized random arbitrarily primed polymerase chain reaction (RAP-PCR) to analyze gene expression in SRB.
  • Performed PCR and genomic sequence analysis to characterize observed chimeric sequences.

Related Experiment Videos

  • Investigated potential genetic mutation mechanisms such as recombination and transposition.

    • Main Results:

    • Chimeric sequences were abundant, comprising 48% of analyzed clones.
    • These chimeric sequences consisted of gene fragments and 16S or 23S rRNA gene sequences.
    • Genomic analysis ruled out genetic mutations as the cause of chimeric sequence formation.

    Conclusions:

    • The formation of chimeric sequences in RAP-PCR is a significant artifact.
    • This observation necessitates careful interpretation of RAP-PCR data, especially when studying differential gene expression.
    • Awareness of this phenomenon can improve the reliability of gene expression studies using RAP-PCR.