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Biotechniques. 2001 Oct;31(4):776-8, 780, 782.

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.

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  • 1Imperial College of Science, Technology and Medicine, London, UK. daron.smith@ic.ac.uk

Abstract

Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.

PMID:
11680707
[PubMed - indexed for MEDLINE]
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