August 1, 2011 (Vol. 31, No. 14)

Higher Throughput and More Sensitive Techniques Keep Workhorse Tool at the Forefront

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.”

Principle of operation of molecular beacon probes: Free molecular beacon probes are nonfluorescent because a quencher moiety is in close proximity to a fluorophore. When the probe sequence in the loop of a molecular beacon probe binds to a target nucleic acid, however, a conformational reorganization occurs that restores the fluorescence of a quenched fluorophore. [Salvatore Marras, UMDNJ]

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.”

Increasing numbers of researchers are adopting qPCR for for gene-expression analysis. While taking the technique in numerous innovative directions, scientists are also working to improve consistency and build confidence in data analysis. [Science Source]

Re-Engineering E. coli

Biophysicist Peter Carr, Ph.D., a research scientist in MIT’s Media Lab’s Center for Atoms and Bits, presented work—qPCR Methods for Re-Engineering Microbial Genomes: Creating a New Genetic Code—in which a novel multiplexed qPCR approach is helping MIT researchers re-engineer the E. coli genome.

The broad project involves investigating methods for effectively modifying genomes. Along with Farren Isaacs, Ph.D., then working in George Church’s Lab at Harvard, Dr. Carr collaborated on MAGE (multiplexed automated genome engineering), a method that simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity.

“We decided to re-engineer the genetic code by rewriting the existence of a given codon throughout the entire genome,” said Dr. Carr. They chose the stop codon Amber and are going through the entire genome of E. coli, which has 314 instances of the codon in the strain being used, and are removing all of them. “To do that all you really need to do is flip one base. We needed a way to validate what we’ve made and also ways of finding the best clones.”

Normally, multiplexed PCR means you are performing up to four PCR reactions in parallel. Each reaction releases a different fluorophore to be detected on a different optical channel. This typically requires a combination of expensive reagents and highly optimized reactions, noted Dr. Carr.

“What we’re doing is mechanistically asking the question: “Which clone is the most modified?” So, imagine we’ve got ten pairs of PCR primers in one reaction tube, and we are querying the genome with primers that either end in G or in A. In other words, they are either a perfect match to the original sequence or to the modified sequence,” said Dr. Carr. “When we are looking at the mutant PCR reactions, all of the ten mutant primers will come up earliest.”

Dr. Carr’s group kept costs down by doing no sample prep. “You don’t hear that very often in qPCR. We use kind of a homebrewed recipe, the cheapest hot start Taq polymerase we can find and cyber green detection. We’re able to do a good job of measuring allele frequencies between 1 percent and 99 percent and doing it with nonoptimized primers, very simple chemistry and very cheap off-the-shelf biochemistry.”

Obtaining a sufficient amplicon size is important in detecting DNA damage. Ziping Zhang, Ph.D., research assistant professor, Texas State University, presented a novel, fast, and precise real-time extra long-PCR (XL-PCR) assay for DNA damage detection based on introducing SYTO-82 to TaKaRa LA Taq™ hot start system as the fluorescence reporter.

“The primary problem solved by RT XL-PCR in this application is that it incorporates XL-PCR into RT-PCR. Traditional RT-PCR is usually good at quantifying short amplicons (50–300 bp). In the present application, it can be used to detect amplicons as long as up to 15 kb. The toughest part of the procedure to get working well is to establish a good RT XL-PCR condition including primer, reporter dye, polymerase, etc. The overall aim is to get constant amplification efficiency for long amplicons,” explained Dr. Zhang.

Dr. Zhang expects the technique to be widely adopted by researchers “to investigate the integrity of DNA in large-scale, for instance, DNA damage detection and DNA repair. It also allows DNA stability comparison from different regions of genomes or chromosomes. It is quite easy and simple, new skills or equipment are not needed. What you need to do is to design a good pair of primers for RT XL-PCR, you can run it on any RT-PCR machine with SYTO-82 detector.”

Currently, this method takes a longer time than normal RT-PCR, but Dr. Zhang expects development of new high-speed Taq shorten the time required.

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