Plant Molecular Biology Reporter

, Volume 23, Issue 2, pp 139–143 | Cite as

Consistent production of cost-effective longSAGE libraries

  • Allison Clare Crawford
  • Jessica White
  • Peter Bundock
  • Giovanni Cordeiro
  • Shane McIntosh
  • Toni Pacey-Miller
  • Lee Rooke
  • Robert James Henry
Commentary

Abstract

Serial analysis of gene expression (SAGE) and related techniques are gaining popularity as tools for exploring expression of plant genes but remain suboptimal because of smaller-than-expected average concatemer sizes. The presence of low-molecular-weight contaminants in high-molecular-weight concatemer fractions reduces the average size of cloned fragments, thereby limiting the viability of high-throughput sequencing methods. Implementation of an additional digestion step to promote formation of linear concatemer fragments appears to reduce the proportion of contaminants indirectly, but with variable results. We explored the effect of initial ditag polymerase chain reaction (PCR) quantity on the average size of cloned concatemers from the greater than 1000-bp fraction. The quantity of PCR material used was found to have a strong influence on the frequency of low-molecular-weight contaminants within this fraction, which has important implications for reducing costs associated with high-throughput sequencing of concatemer clones.

Key words

barley cost-effective gene expression modification protocol transcript abundance transcriptome 

Abbreviations

PCR

polymerase chain reaction

RL-SAGE

robust longSAGE

SAGE

serial analysis of gene expression

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Copyright information

© Springer 2005

Authors and Affiliations

  • Allison Clare Crawford
    • 1
  • Jessica White
    • 1
  • Peter Bundock
    • 1
  • Giovanni Cordeiro
    • 1
  • Shane McIntosh
    • 1
  • Toni Pacey-Miller
    • 1
  • Lee Rooke
    • 1
  • Robert James Henry
    • 1
  1. 1.Grain Foods CRC, Centre for Plant Conservation GeneticsSouthern Cross UniversityLismoreAustralia

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