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Oct 15, 2009 (Vol. 29, No. 18)

qPCR Faces Growing Pains Head-On

Establishment of Standards and Evolution of Methodologies Help the Field Mature

  • Unique Twist with RNase H

    Click Image To Enlarge +
    Blocked/cleavable primers that improve PCR specificity (Integrated DNA Technologies)

    Integrated DNA Technologies (IDT) has developed a new system for detecting nucleic acids in complex samples. According to Mark Behlke, CSO, the company is “utilizing a novel system that employs PCR with modified primers that have an internal RNase H2 cleavage site. RNases H are endonucleases that specifically hydrolyze the RNA strand in an RNA/DNA hybrid.

    “In this system, a cleavable linkage (RNA base) is positioned near the 3´-end of the DNA primer which is blocked at the 3´-end and cannot function as a primer without cleavage. After the primer hybridizes to its target, the terminal blocking group is removed by the RNase H2 enzyme and amplification occurs. The hybrid is sensitive to correct base pairing so that unblocking is inhibited by the presence of a base mismatch near the location of the cleavage site.”

    Dr. Behlke reported that the technology is especially useful for enhancing specificity such as for high-homology gene-expression studies, rapid SNP analysis, and allele discrimination.

    Presenting an example that employed SYBR™ Green in the PCR assay, Dr. Behlke described experiments using human or rat cDNA with primers specific for the human HRAS gene, a member of the Ras oncogene family.

    “The PCR reactions were run for 60 cycles. Despite the relatively large differences in sequence between the rat and human genes, a false-positive signal was generated in rat cDNA using human-specific primers. When we converted the identical sequences to the blocked primer design and used the same target cDNA samples, the blocked sequences showed the same true positive signal in human cDNA, but did not give any false amplification in rat cDNA. Although we used SYBR Green, it is also possible to use blocked primers labeled with a fluorophore and a quencher. This alternative format permits multiplexing.”

    The company expects to release the technology in 2010. It is currently performing beta testing, preparing papers for journal submission, and seeking partners for applications for in vitro diagnostics.

    The remarkable progress made in the field of quantitative PCR also has generated some growing pains. As researchers continue to refine and tackle problems, most remain optimistic that the maturing of qPCR technology will involve becoming more rapid, user friendly, capable of higher throughput, and cost effective. 

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