Better Tumor Diagnostics
A powerful PCR technique for use in profiling cancer tumors—LNA-Enhanced Real-Time Ice-COLD-PCR and High Resolution Melting for Ultra-Sensitive Detection of Low-Level Lung Cancer Resistance Mutations—was presented by Mike Makrigiorgos, Ph.D., director biophysics laboratory and medical physics division, Dana Faber Cancer Institute, Harvard Medical School. The problem addressed by Ice-COLD-PCR and COLD-PCR (the original advance that lead to Ice-COLDPCR) is that clinical cancer samples are never pure, but they are always mixed with normal, wild-type cells.
“Ice-COLD-PCR provides a unique method for detection of low-level mutations—for example, mutations below 5 percent mutant to wild-type alleles—because it magnifies subtle mutations during PCR amplification such that following PCR they can easily be identified,” explained Dr. Makrigiorgos.
Ice-COLD-PCR, an advance over COLDPCR, stands for improved and complete enrichment of mutations via co-amplification at lower denaturation temperature PCR. A full explanation was published by Dr. Makrigiorgos and colleagues in Nucleic Acids Research, 2011, Vol. 39, No. 1.
In brief, it combines elements of fast COLD-PCR and full COLD-PCR as explained in this excerpt from the paper: “… to enrich all mutation types, Ice-COLDPCR employs a reference sequence (RS) of a novel design; the RS is engineered such that (i) it matches the WT-sequence of the antisense strand; (ii) PCR primers cannot bind to it; and (iii) it is phosphorylated on the 3'-end so that it is nonextendable by the polymerase.
When incorporated into PCR reactions in excess relative to the template, the RS binds rapidly to the amplicons. At a critical denaturation temperature, the RS:WT duplexes remain double-stranded, thereby inhibiting selectively the amplification of WT alleles throughout the thermocycling. Conversely, the RS:mutant duplexes are preferentially denatured and amplified.”
“The unique aspect of COLD-PCR is that there is no need of a priori knowledge as to the type and position of a mutation. All mutations are magnified irrespective of their position on the sequence. Accordingly, following Ice-COLD-PCR one may apply direct sequencing to the PCR product to identify the type and position of the mutation,” said Dr. Makigiorgos.
While reliable NGS for DNA with highprevalence tumor somatic mutations has been demonstrated, Dr. Makigiorgos noted the required “depth” of sequence interrogation remains a problem, and detection of low-prevalence somatic mutations at levels below ~2–5% in tumors with heterogeneity, stromal contamination, or in bodily fluids is fraught with false-positives irrespective of coverage.
“We intend to establish massively parallel Ice-COLD-PCR to enrich mutant sequences prior to their screening via nextgeneration sequencing, thus enabling ultradeep NGS while also retaining accuracy, reliability, and high-throughput capability,” said Dr. Makigiorgos.
The new approach has already been adopted by several groups worldwide and is being used for diverse applications that include: cancer-based molecular diagnosis, prenatal diagnosis, plant/crop genetics, and infectious diseases.
Ice-COLD-PCR requires special DNA constructs to work, and Dr. Makigiorgos said the constructs should soon be available commercially, “as Dana Farber Cancer Institute has licensed a portion of the rights to Ice-COLD-PCR to Transgenomic, which is developing Ice-COLD-PCR assays for specific clinically relevant genes.”