Enrichment of low-abundance gene mutation by a combined polymerase and ligase chain reaction

Abstract

Detection of low-abundance gene mutations or minority alleles in clinical samples is important and challenging in fields of tumor, infectious diseases, noninvasive prenatal diagnosis, and forensic science. The key to solving this problem is the selective enrichment of the low-abundance gene fragments. In this study, a combined polymerase and ligase chain reaction system based on conventional polymerase chain reaction was developed for the first time by introducing a heat-resistant DNA ligase and a pair of ligation primers that target the mutant site. Three EGFR gene mutations (L747_S752 del, G719A, and T790M) were used as the target mutations. Both artificial mixed samples containing 1% of 1 of the 3 EGFR gene mutations and tumor samples were used to evaluate the feasibility of the proposed new method. Sanger sequencing results showed the coexistence of the wild-type and mutant alleles when the amplification product was obtained by the combined polymerase and ligase chain reaction. The novel method based on the combined polymerase and ligase chain reaction has good performance in the detection of the 2 EGFR mutations, as the commercial kit. The novel method could be used to effectively inhibit the amplification of the wild-type gene fragment in the sample and selectively amplify the low-abundance gene fragment with a mutant site, allowing the mutation to be subsequently detected more effectively, accurately, and reliably. By using this novel method, the mutant gene present at an initial content as low as 1% could be effectively amplified and accurately detected.

Keywords: combined polymerase and ligase chain reaction; gene enrichment; heat-resistant DNA ligase; low-abundance gene mutations; minority alleles.

Figure 1.

Schematic diagram of the novel method for enriching low-abundance genes. The * on the mutant gene in the diagram represents the mutation site; The left arrows on gene templates represent the amplification primers and their extension direction; L and R represent the ligation primers (the solid dots represent the phosphorylation modification); The triangle represents DNA polymerase, the circle represents DNA ligase; The tilted right end of the L ligation primer on the mutant gene template in the annealing phase indicates that it does not match the mutation site. The dashed arrows extended from the amplification primer in the expansion phase represent the extension of the DNA strand; The inclined dashed arrow in the upper right corner of the ligation primer in the extension phase indicates that the primer is detached from the mutant gene template; The long ligation primer is formed by the ligation primers L and R through DNA ligase.

Figure 2.

Sanger sequencing profiles for L747_S752 deletion mutation site. (A) corresponding to the amplification fragment of the sample control or the reaction control, and (B) corresponding to the amplification fragment of the mixed clones sample with L747_S752 deletion mutation fragment. The red circle indicates the mutation site, and the red arrow indicates the mutation information.

Figure 3.

Sanger sequencing profiles for the T790M point mutation site. (A) corresponding to the amplification fragment of the sample control or the reaction control, and (B) corresponding to the amplification fragment of the mixed clones sample with T790M point mutation fragment. The red circle indicates the mutation site, and the red arrow indicates the mutation information.

Figure 4.

Sanger sequencing profiles for the G719A point mutation site. (A) Corresponding to the amplification fragment of the sample control or the reaction control, and (B) corresponding to the amplification fragment of the mixed clones sample with G719A point mutation fragment. The red circle indicates the mutation site, and the red arrow indicates the mutation information.