Protocol

PCR Protocols

Volume 687 of the series Methods in Molecular Biology pp 265-274

Date:

Application of Blocking Oligonucleotides to Improve Signal-to-Noise Ratio in a PCR

  • Hege VestheimAffiliated withDepartment of Biology, University of Bergen
  • , Bruce E. DeagleAffiliated withDepartment of Biology, University of Victoria
  • , Simon N. JarmanAffiliated withAustralian Antarctic Division, Australian Marine Mammal Centre

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Abstract

“Universal” or group-specific PCR primers have a tendency to predominately hybridise with the common sequences in samples with mixed templates. The result is that the rarer sequences are seldom retrieved by cloning or sequencing. The use of a blocking oligonucleotide (oligo) designed to specifically prevent amplification of dominant or unwanted DNA templates is an easy way to improve the amplification of rarer sequences. Here, we describe the different types of blocking principles and the different types of blocking oligos and give guidelines and examples of their application.

Key words

Blocking probes Blocking primers Blocking oligonucleotide Competitive probes C3 spacer, peptide nucleic acids Locked nucleic acids Allele-specific competitive blocker PCR Enhanced amplification of rare sequences