A tailored LNA clamping design principle: efficient, economized,
specific and ultrasensitive for the detection of point mutations
Abstract
In the development of personalized medicine, the ultrasensitive
detection of point mutations that correlate with diseases is important
to improve the efficacy of treatment and guide clinical medication. In
this study, locked nucleic acid (LNA) was introduced as an amplification
suppressor of a massive number of wild-type alleles in an amplification
refractory mutation system (ARMS) to achieve the detection of
low-abundance mutations with high specificity and sensitivity of at
least 0.1%. By integrating the length of clamp, base type, number and
position of LNA modifications, we have established a “shortest length
with the fewest LNA bases” principle from which each LNA base would
play a key role in the affinity and the ability of single base
discrimination could be improve. Finally, based on this LNA design
guideline, a series of the most important single point mutation sites of
epidermal growth factor receptor (EGFR) was verified to achieve the
optimal amplification state which as low as 0.1% mutation gene
amplification was not affected under the wild gene amplification was
completely inhibited, demonstrating that the proposed design principle
has good applicability and versatility and is of great significance for
the detection of circulating tumor DNA.