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UCSD Scientists Optimize PCR-Based Method for Detecting DNA Deletion Mutations

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By Ben Butkus

A team of computational biologists and cancer researchers from the University of California, San Diego, has optimized a PCR-based method for detecting deletion mutations caused by somatically acquired DNA rearrangements, according to a recently published research paper.

As a result of the work, the method — dubbed primer approximation multiplex PCR, or PAMP — may be able to serve as the basis for diagnostic tests for cancers characterized by these mutations, one of the study's authors said this week.

The PAMP method got its start at UCSD's Department of Computer Science and Moores Cancer Center, where Yu-Tsueng Liu and Dennis Carson developed the technique to map the breakpoints in deletion mutations, which are increasingly implicated in a number of cancers.

One of the major difficulties in detecting such deletions, however, is that DNA regions from early-stage tumors are often drowned out by copious amounts of normal DNA. This makes sequencing-based detection impractical due to the enormous depth of sequence coverage needed; while PCR-based detection is difficult because the boundaries of rearrangement mutations are not conserved across individuals and may vary by hundreds of kilobases.

In the PAMP method, researchers "tile" a genomic region of interest with forward/reverse primer pairs, all of which are placed in one or more multiplex tubes with the sample DNA. The primers are spaced in such a way that a deletion at any boundary will bring a primer pair close enough together for amplification, the product of which is hybridized to a set of probes and detected on an array. Using the method, researchers can confirm a deletion event and resolve its boundaries to within one kilobase.

In a 2007 paper published in PLoS One, Liu and Carson described PAMP for the first time and used it to identify the genomic breakpoints in cancer-derived CDKN2A gene deletions amidst a background of more than 99 percent normal DNA.

But even in that paper, the researchers conceded that scaling up PAMP and using it to detect deletions in a diagnostic capacity would require bioinformatics support. As such, they brought in additional UCSD computational biologists on the project, one of whom was Vineet Bafna.

Bafna this week explained in an interview with PCR Insider that the first challenge of designing PCR primer sets for PAMP is that the technique requires enough primer pairs to detect alterations within a very large range of base pairs — as many as 500,000. Furthermore, the forward/reverse primer pairs must be able to completely cover the region of interest.

"You have to choose these primers out of all possible primers, and in this region you might find 5,000 good primers," Bafna said. "If you put them individually in the reaction they would have the right properties and help amplify."

It would be near impossible to test all 5,000 primers, however, so the researchers must narrow their choices down to something more manageable, around 500 primers optimized for the method.

"The primers must be equally spaced," Bafna said. "They must tile the region in such a way that no matter where the deletion happens, there is a pair of primers no more than 2,000 base pairs apart. That's the part that we really want. Suppose you had a deletion, and there was no primer nearby. Then you miss that deletion and miss the diagnosis. A false positive is not so bad … but false negatives are really bad."

Complicating matters is the fact that primers must be selected in such a way as to avoid the formation of primer-dimers, just one of which has the potential to destroy all other signals in a multiplexed reaction.

"We take every pair of primers and computationally test if they can stick to each other," Bafna said. "And then, from the 5,000 primers, we select the 500 such that no pair will stick to each other and they tile the region properly."

But again, "because there is no way to test all possible subsets … that's where our computation comes in," Bafna said. The approach "takes all these constraints where the primers must be equally spaced, must not stick to each other, and then it tries to find the best 500 primers that satisfy all these constraints."

Bafna and UCSD researchers Ali Bashir, along with Liu, Carson, and other contributors, described their computational method in a paper published in 2007 in Bioinformatics. In that paper, they detailed how they described the necessary primer constraints as a combinatorial optimization problem, and designed algorithms for PAMP primer design based on simulated annealing and integer programming.

"But there are even some problems with that," Bafna said. "There were some technical difficulties. The protocol would not work in many cases in the original paper, but now we've corrected that." The researchers updated their approach in a paper published last week in the Journal of Computational Biology.

For instance, because the researchers detect primer amplification by hybridization against a probe, oftentimes "there are two possible pairs of primers that could amplify, but the probe is located in such a way that it works for one pair but it doesn't work for the other pair," Bafna said. "And the pair it doesn't work for is the one that gets amplified. To address this issue, the researchers employed what they termed an alternating multiplexing strategy.

In addition, the researchers discovered that their previous method of placing all 500 primers in one mix too frequently resulted in primer-dimer formation. To work around this, they were able to further reduce the number of optimized reactions in each mix using a graph coloring model.

As described in the JCB paper, Bafna and colleagues were able to use their computational method to custom design an assay for genomic lesions in several cancer cell lines associated with a disruption in CDKN2A. They were able to detect the deletion breakpoint in all cell lines, even when the region had undergone multiple rearrangements.

Bafna said that the results are very promising for developing the PAMP technique into a test for early diagnosis and monitoring of cancer. "But now we need to test this in original tumor data, and lots of it," he said. "But we are not currently funded to do that, so the project is a bit stalled right now. It is still active, but we haven't been able to do anything since this last publication."

Nevertheless, in the meantime, "there are many other things we can do from a technical perspective," Bafna said. For instance, the group is exploring how it might be able to combine its method with technologies that enable highly multiplexed PCR, such as those offered by RainDance Technologies or Fluidigm.

"They offer a way of multiplexing, so one thing we are thinking about is combining this approach with some of these next-gen amplification techs to try and get some results that way," he said. "Anything that they do, the computation method could definitely help. And then the PAMP itself for detecting deletions in cancer is a novel way of applying PCR. So these three ideas can be combined in many different ways."

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