Finnish researchers have created a PCR protocol involving multiple heat pulses in the extension step to enable amplification of extremely GC-rich sequences and are optimizing the method to diagnose a number of repeat expansion diseases.
The Helsinki University Central Hospital is currently using an in-house version of the method, called HPE-PCR, to diagnose one such disease, type 1 myotonic dystrophy. The researchers, who are affiliated with the University of Helsinki, have also formed a company called Expression Analytics to further develop diagnostic tests for other repeat expansion disorders like fragile X syndrome and amyotrophic lateral sclerosis.
Additionally, the group is interested in optimizing the heat pulse protocol to pre-amplify DNA for next-generation sequencing, according to Jakob Stenman, a researcher at the Institute for Molecular Medicine Finland and one of three authors of a study detailing the method that was published online last week in Analytical Chemistry.
In the study, the group compared HPE-PCR to other recently published methods that focus on adapting the annealing step and showed that HPE was able to amplify all CGG repeat expansions in five fragile X cell lines, as well as extremely GC-rich non-repetitive segments of two other genes.
PCR amplification over GC-rich and/or long repetitive sequences is challenging because target sequences may denature incompletely, re-anneal, and self-anneal to create thermostable structures. Several methods for improving GC-rich amplification have been developed, either through combining special reagents to improve amplification, or optimizing cycling protocols, usually in the annealing step.
However, in spite of these improvements, amplification of long, extremely GC-rich and/or repetitive fragments "still remains challenging and in some cases impossible," Stenman and co-authors wrote in their study, because secondary structures with high melting temperatures remain a major obstacle.
"The really important thing that is different with this heat pulse extension is that we're focusing on the extension step of PCR, whereas almost all other techniques [for amplifying GC-rich regions] focus on the annealing step," Stenman told PCR Insider this week.
He explained that the group, led by Arto Orpana, a chemist at the Laboratory for Molecular Genetics at HUCH, came up with the idea of heat pulsing in the extension based on computer simulations of how long, GC-rich PCR products fold into themselves at different temperatures.
"When you look at those secondary structures they're different for every temperature, so if you increase the temperature, the molecule will actually unfold and refold in a new configuration," Stenman said.
"This brought up the idea that by continuously moving the temperature up and down — actually as quickly as we can make the machine work — then every time the molecule takes a new configuration there is an opportunity for the polymerase to push forward a few bases."
Not only can this approach amplify the GC-rich sequences, Stenman explained, but it also minimizes amplification bias between sequences of different lengths.
In their study, the Helsinki group detailed the performance of the HPE-PCR method on a panel of fragile X cell lines containing different expansions with known numbers of repeats, from 23 to 940 CGG copies.
The HPE extension begins with gradual heating to a basal extension temperature between 72° and 80° C, the authors reported. Then the temperature is raised in multiple pulses to a peak between 80° and 90°, quickly dropping back to the basal temperature between pulses. In the study, 14 pulses were sufficient for all tested sequences, the authors wrote, though differences in length, sequence, and GC-content could require different numbers of pulses.
The HPE protocol enabled specific amplification of all expansion lengths in the fragile X samples, with the longest containing 940 CGG repeats and corresponding to a 2.8-kb segment of 100 percent GC content, the authors reported.
The team also tested the cell lines using conventional PCR with several elevated extension temperatures and found that while some temperatures showed improvement, none were able to amplify the largest 940-repeat expansion. Conventional cycling also produced amplification bias, the authors reported, while the HPE-PCR method successfully amplified both the large expansions and the wild-type unexpanded alleles.
The researchers also used the same reagents to evaluate several other recently reported protocols such as slowdown PCR and hotstart and touchdown PCR.
Stenman said that all of these compared poorly with HPE-PCR. Although the researchers did not do any "extensive optimization" with these other methods, he said, "really there are no reports of anyone who has been able to amplify 3 kilobases of 100 percent GC without a lot of special reagents [with these methods], so we know [our] method does something that hasn't been possible [before.]"
HPE-PCR, as the group describes in their paper, takes several times longer than standard PCR — between four and six hours, but Stenman said the group is working to bring that time down.
Also, Stenman said, there are even longer repeats, up to tens of kilobases, that the method can not yet amplify. "That's something we're working with because we obviously want to be able to amplify anything and we're not quite there yet."
As described, the HPE-PCR protocol uses standard PCR reagents in contrast to more reagent-focused methods for fragile X syndrome, like Asuragen's AmplideX FMR1 PCR kit, which received a CE Mark in Europe earlier this year and currently has research-use-only labeling in the US (PCR Insider, 10/6/11).
Stenman said one approach doesn't exclude the other, and suggested that adopting specialized reagents may help his group increase the abilities of the HPE method to amplify even greater repeats.
"We haven't extensively tested other reagents, but I can imagine it would be a way to further the method, to combine it with other techniques that have been published." However, he said, in Asuragen's case "they didn’t really disclose what their special reagents were, so it's difficult [to see if there could be a possibility of combining.]"
Even though the HPE protocol takes longer than standard PCR, Stenman said the team believes its ability to amplify different length repeats without bias holds promise for use as a diagnostic for repeat extension diseases.
Stenman said that the group started with type 1 myotonic dystrophy, because the disease's expansions tend to be shorter and are not "100 percent GC," which made it an easier first target.
"Now that we've continued optimizing the technique, we realize we can easily amplify very difficult templates also, so that's why we're including other genes," he said.
In the near future, Stenman said the group believes they will be able to use the method to diagnose fragile X syndrome and ALS, which has only recently been shown to involve repeat expansion. Additional targets could include type 2 myotonic dystrophy as well as Huntington's disease, he added.
Fragile X is the most common form of inherited mental retardation, and arises from expansion of a CGG trinucleotide sequence in the 5'-untranslated region of the FMR1 gene.
With diseases like DM1, and maybe fragile X, Stenman said, Expression Analytics could be perhaps only one or two years away from commercializing diagnostic tests. But he said the company doesn't yet have a "global partner" and thus has not solidified plans for bringing an HPE-PCR test to market.
Additionally, he said the group is working to develop pre-amplification protocols for next-generation sequencing that would take advantage of the method's ability to amplify without wild-type bias.
"Currently, we see that semiconductor-based sequencing will be an extremely cheap detection method within a foreseeable future," he said. "But at the moment, practically all sequencing methods are still dependent on pre-amplification and if you have bias during pre-amplification, it doesn’t matter how good your sequencing is, you'll still have bias in the assay."
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