Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples

BMC Genomics. 2010 Jul 2;11:413. doi: 10.1186/1471-2164-11-413.

Abstract

Background: The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo.

Results: In this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background.

Conclusions: Our results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • DNA, Complementary / biosynthesis*
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Gene Library
  • Isomerism
  • Molecular Sequence Data
  • Moloney murine leukemia virus / enzymology
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism*
  • Plant Cells
  • Plants / genetics
  • Polymerase Chain Reaction / methods*
  • RNA / metabolism*
  • RNA-Directed DNA Polymerase / metabolism
  • Sequence Analysis, DNA / methods*

Substances

  • DNA, Complementary
  • Oligonucleotides
  • RNA
  • RNA-Directed DNA Polymerase