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Researchers from Italian Cancer Institute Develop PNA 'Clamping' PCR Method for SNP Detection


By Ben Butkus

LONDON – A team of biologists from an Italian cancer research institute has developed a PCR method that can detect any SNP within genomic DNA inexpensively and with comparable or better sensitivity to existing methods, one of the research team members said at a qPCR conference held here this week.

The technique, dubbed peptide nucleic acid-mediated competitive-PCR clamping, could serve as an inexpensive new assay for routine diagnostic applications and pharmacogenomic assays, and the team has already optimized tests for several important genetic mutations in the hematology and oncology fields.

In a presentation at Oxford Global's qPCR, Next-Generation Sequencing, and Analytical Genomics Congress, Raffaelle Di Francia, a scientist from the Laboratory of Molecular Hematology at Naples' National Institute of Tumours Fondazione G. Pascale, discussed the development of the technique and presented data demonstrating its ability to detect a mutation in the JAK2 gene associated with myeloproliferative disorders.

At the crux of the method are PNAs, or peptide nucleic acids, which have structural similarity to DNA or RNA but with a different chemical backbone structure. PNAs are used in a number of molecular biology techniques, such as PNA-assisted rare cleavage and as probes in Förster resonance energy transfer or quartz crystal microbalance nucleic acid binding assays.

Di Francia and colleagues have adapted the molecules to quantitative real-time PCR assays by designing high-specificity PNA probes that "clamp" the wild-type allele, thereby preventing PCR synthesis of those targets and enhancing PCR synthesis of the mutant allele — essentially creating a competitive binding assay that can promote highly sensitive PCR amplification of only selected mutant alleles in a large excess of wild-type nucleic acids.

To test the method, the group performed a validity test for the V617F mutation in the JAK2 V617F mutation in 250 total white blood cell samples taken from patients with myeloproliferative disorders such as polycytemia vera and essential thrombocytemia, achieving a sensitivity of between 95 and 99 percent, Di Francia said.

A number of other assays for detecting the JAK2 V617F mutation — such as PCR plus sequencing, allele-specific oligonucleotide PCR (alone and followed by capillary electrophoresis), amplification refractory mutation system PCR, and allele-specific PCR plus locked nucleic acid probes — have been evaluated in the literature

And while PNA clamping PCR demonstrated sensitivity comparable to the published sensitivities of most of these methods, Di Francia and colleagues believe that it is more time- and cost-effective.

Also, in a smaller-scale experiment, they compared the ability of their method to detect the mutation to both ARMS-PCR and ASO-PCR. While PNA clamping PCR detected 100 percent of mutated and wild-type genes, the ARMS-PCR technique detected about 60 percent of mutated and 88 percent of wild-type sequences; while AS-PCR detected about 88 percent of mutated and 100 percent of wild-type sequences. All samples were confirmed by DNA sequencing, Di Francia said.

Di Francia said that the method could be performed on either genomic DNA or cDNA sources in a single-step reaction; and, if incorporated into routine diagnostic or pharmacogenomic assays, could clock in at a cost of about €5 ($6.80) per reaction.

In an interview with PCR Insider following his presentation, Di Francia said that the method is patent-pending in Italy, but that the group has not yet pursued intellectual property rights elsewhere.

He also said that the group has optimized several different assays based on PNA clamping PCR in the areas of hematology and oncology. Examples include an assay for a gene mutation in follicular lymphoma; and a pharmacogenomics panel of the BCR-ABL gene in imatinib-resistant leukemic clones.

The institute is seeking partners interested in further developing the method for various applications, Di Francia said.

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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