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

qPCR's Steady Advance Delivers New Applications

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

  • 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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