By Ben Butkus
Scientists from Korea's Samsung Medical Center have developed a sequence-enrichment technique for identifying trace mutant DNA sequences in a high background of normal DNA, according to research published last month.
The method, called mutant enrichment with 3'-modified oligonucleotides, or MEMO, can be used in tandem with quantitative real-time PCR, high-resolution melt curve analysis, or downstream Sanger sequencing to detect common cancer mutations with a high degree of sensitivity and specificity, and is cheaper and easier to use than existing methods, according to the researchers.
Despite the technique's potential cost savings, however, it may not provide the same degree of enrichment for downstream PCR analysis as current methods, and may not be able to enrich unknown mutations in longer sequence stretches, according to one expert.
The researchers from Samsung Medical Center, an independent research institute and teaching hospital of the Sungkyunkwan University School of Medicine, described the MEMO method in a paper published in October in the Journal of Molecular Diagnostics.
The technique, they wrote, is similar to PCR clamping techniques that use peptide nucleic acids or locked nucleic acids — the rights of which are owned by Life Technologies and Exiqon, respectively — to increase probe specificity and binding strength.
However, in MEMO the PNA or LNA is replaced by 3'-modified oligonucleotides that feature an extension-inhibiting compound on their 3' ends and are added to a PCR reaction mixture along with two generic primers.
The blocking primer encompasses the target mutation site and complements the wild-type sequences. One of the two generic primers overlaps with the blocking primer by several bases, neighboring the target mutation site and thus competing with the blocking primer, the researchers wrote.
In turn, the DNA binding of the blocking primer "dominates for wild-type sequences, whereas its affinity for mutant sequences is markedly reduced due to mismatches," the researchers wrote. "The loss of competition of the blocking primer enables selective amplification of mutant sequences by the generic primer pair."
As described in the JMD paper, the Korean researchers evaluated the ability of the MEMO method to detect common cancer mutations in the EGFR, KRAS, BRAF, TP53, JAK2, and NPM1 genes, and observed sensitivities ranging from 10-2 to 10-6 when combining the technique with downstream Sanger sequencing.
According to the researchers, this sensitivity depends on the properties of the primers and the experimental conditions, as well as the nature of the mutation and its surrounding sequences.
In addition, although MEMO can potentially be coupled with subsequent quantitative real-time PCR and melting curve analysis, the researchers found that the technique's sensitivity suffered in these instances.
"Although MEMO-PCR and Sanger sequencing showed sensitivities up to 10-6, MEMO-PCR and melting curve analysis showed ambiguous patterns in samples with very low mutant concentrations (from 10-6 to 10-4)," the researchers wrote. "This might be attributed to the fact that the resolution of melting curve analysis is affected by many factors including template or final DNA concentrations."
Nevertheless, the authors claimed that at the very least, MEMO combined with Sanger sequencing resulted in sensitivities similar to those of PNA- or LNA-mediated methods, and the MEMO probes "are much less expensive and are easy to design."
It is unclear how much less expensive MEMO probe design would be compared to PNAs or LNAs. The Korean researchers did not provide cost details in their paper, and the corresponding author could not be reached for comment.
Mike Makrigiorgos, an associate professor and director of medical physics and biophysics at Dana-Farber Cancer Institute and inventor of a competing enrichment method — co-amplification at lower denaturation temperature, or COLD-PCR — told PCR Insider this week that although the MEMO technique was "elegant," it seemed to be a minor modification of concepts that have existed for a while.
"PNA [and] LNA probes that operate on the same principle as MEMO have been known for a while," Makrigiorgos said. And while he agreed that PNA/LNA probes are more expensive than MEMO probes, "one would expect that they provide a better enrichment of mutant DNA during PCR. Finally, high-resolution melting techniques already use unmodified oligonucleotides … similar to MEMO … to identify base changes and SNPs following PCR."
Makrigiorgos also noted that COLD-PCR and an improved version recently developed in his lab called Ice-COLD PCR have an advantage over MEMO and techniques such as PNA- and LNA-mediated clamping because "they uniquely enable enrichment of unknown mutations at any position of longer sequences," for example, of 150 to 200 basepairs, "and hence match well with downstream sequencing technologies. Techniques like MEMO and LNA/PNA essentially address mutations at known positions of sequences up to 20 bases long."
Indeed, the Korean researchers noted in their paper that current limitations of MEMO include the fact that "only mutations in a known target region (within the coverage of blocking primers) can be enriched, and sensitivities may vary according to different base substitutions in the same target regions."
Even so, the researchers believe that MEMO has the potential to provide a cheap and easy alternative in situations where "minority alleles of clinical significance are present and sensitive detection is required."
Besides cancer mutation detection, the authors suggest that the technique can be useful for identifying variant strains in infectious diseases, detecting minor mutant alleles in patients with low-level somatic mosaicism or mitochondrial heteroplasmy, and characterizing fetal mutations from maternal plasma samples.
Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.