The Study Of A Novel Low-Abundance Unknown Gene Mutation Enrichment Method Based On Locked Nucleic Acids

Gene mutation is a naturally occurring phenomenon, which is the engine of evolution. In most cases, however, gene mutations often have negative effects on creatures, which lead to sickness and even death. Therefore, detection of gene mutations in the kinds of biological and medical samples is of theoretical and practical significance. So far, many detection methods have been developed. Nevertheless, the proportion of mutation in samples is very low in many cases. Gene variants often coexist with their wild-type counterparts. Also, wild-type DNA usually tend to be much more than mutant DNA, making it difficult to detect and identify minority alleles. Yet it is very important to identify such’needles in a haystack’mutations in many regards, especially for early cancer detection, determining personalized treatment and therapy, and noninvasive prenatal diagnosis.In 2011, a novel unknown gene mutation enrichment platform named ice-COLD-PCR was reported by Makrigiorgos GM and his colleagues. This method introduces a long single strand of synthetic DNA named reference sequence (RS) into the PCR system, which is a little shorter than the amplicon and matches the WT-sequence of the antisense strand. When incorporated into PCR reactions in excess relative to the template, the RS binds rapidly to the amplicons. At a critical denaturation temperature, the RS mutant duplexes are preferentially denatured and amplified. By using a WT-specific RS, all variants can be effectively amplified, regardless of mutational type and position. It has high selectivity, simple in performance. And more importantly, it can be used combined with almost all existing downstream analysis methods with no additional cost, time, and labor. Ice-COLD-PCR is considered as a paragon of low-abundance unknown mutation enrichment methods. However, there is still some disadvantages and room for improvement.On the one hand, because RS is about 30 nt shorter than template, the melting temperature of RS:WT duplex (Tm2) is often a little lower than that of the WT template (Tm1).Therefore, when denatured at the critical denaturation temperature (Tc), which is a little lower than the melting temperature of RS:WT duplex, the antisense strands separated from the RS:variants duplexes may be bind with the sense strands of templates again, which plays great negative effects on the efficiency of mutant enrichment.On the other hand, ice-COLD-PCR was developed based on the phenomenon that single base mismatch will lead a decrease in melting temperature. Because in many cases, the variations of natural DNA melting temperature are very slightly, the extent of differential amplification of ice-COLD-PCR to variants and wild type gene is relative low, which lead to high requirements for the temperature control module of the thermal cycler.To overcome these shortcomings, in this work, the technology of locked nucleic acids (LNA) was incorporated with ice-COLD-PCR, and a novel low-abundance unknown mutation enrichment method named LNA-COLD-PCR was proposed. Unlike natural nucleosides, LNA nucleosides are a class of nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2’-O atom and the 4’-C atom. The bridge "locks" the ribose in the 3’-endo (North) conformation, which is the ideal conformation for Watson-Crick binding and often found in the A-form duplexes. This unique structural feature allows that LNA oligonucleotides possess extremely high binding affinity to its complementary DNA and RNA and an superior ability of single base mismatch discrimination relative to natural nucleic acids.Referred to the original paper of ice-COLD-PCR, in this work, several LNA blocking sequences (BS) were successfully designed and synthesized, and a series of standard samples were also prepared. Through optimizing several parameters such as blocking sequence concentration, critical denaturation temperature, template concentration, and other important parameters, LNA-COLD-PCR platform was successfully constructed. By employing LNA as blocking sequence, LNA-COLD-PCR realized Tm1(template)< Tc< Tm2(BS:WT) and solved the first problem mentioned above. Furthermore, the second problem was also alleviated to a certain extent, due to the properties of LNA. Generally, our LNA-COLD-PCR is predominant in differential amplification compared with ice-COLD-PCR. Moreover, it shows no needs of high quality temperature control module for PCR instruments. Even performed on ordinary thermal cycler, and combined with Sanger sequencing, our LNA-COLD-OCR enabled enrichment of all types of unknown mutations and unambiguous identification of low-abundance (down to 3%) mutations, outperforming COLD-PCR and ice-COLD-PCR.