After more than 25 years of use, PCR remains a workhorse in molecular biology. New applications, new reporter chemistries, and advancing microfluidics will usher in higher throughput and more sensitive PCR techniques, believes Salvatore Marras, Ph.D., and professor, Public Health Research Institute, University of Medicine and Dentistry of New Jersey, and a speaker at Select Biosciences' recent “Advances in qPCR” conference.
“I think the future will include microfluidics where you'll probably do more than 3,000 or 10,000 PCR reactions simultaneously in a microwell,” said Dr. Marras, whose own talk—The Bright and Dark Sides of Fluorescent Nucleic Acid Hybridization Probes—surveyed the broad landscape of probe design and selection of appropriate fluorescent reporter chemistries.
Given the variety of choices available, he said, “I think people put too much emphasis on the intensity of the particular signal during qPCR when it's actually more important to ensure you're able to determine how many copies of the target were present in the sample. There are chemistries that produce signals two or three cycles earlier than other chemistries and as long as you can detect one copy with any available chemistry—such as a Molecular Beacon or Scorpion primer or a TaqMan probe—that's what you want to use. Of course it is important to develop a signal that is significant above the background.”
Looking ahead, Dr. Marras said, “People would like to go to higher throughput assays and higher multiplex assays. So rather than five or six targets to be detected at one time, maybe it will go to 10 or more different targets. This will require new types of fluorophores and new quenchers will probably be developed as well.”
One persistent challenge with qPCR, he explained is “you are able to identify one single copy of particular target chain but the question is: Can you isolate that particular copy out of the sample? To do this, the development of real-time PCR will probably go hand-in-hand with new expression technology that uses a minimum amount of sample to retrieve that copy necessary for you to make the diagnostics.”
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.”