Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene

Abstract

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…111,112 In comparison to the heating, proteinase K digestion and SDS treatment methods, usage of the commercial DNA clean-up kit produced only the target band but in a much lower band intensity. 37,109,111 In addition, as an alternative method, centrifugation (3 minutes) to pellet RPA proteins showed equivalent performance to the heating method (65°C for 10 minutes). 110 As with lateral flow strip detection, direct usage of RPA amplicons is possible, but it is recommended to dilute the amplicons with the running buffer (e.g.…”

“…Most published RPA papers have applied amplicon lengths between 100 and 250 nucleotides, which usually incur fast and efficient amplification. However, shorter amplicons (79 nucleotides; 37 94 nucleotides [38][39][40] and longer amplicon up to 1500 nucleotides 6 and 1941 nucleotides 41 have also been reported. Unlike PCR, the length of RPA primers is relatively long (a recommended minimum of 30 nucleotides, but typically between 32 and 35 nucleotides).…”

“…Shorter PCR primers (typically between 18 and 25 nucleotides) can also be used in the RPA reaction but may decrease the reaction speed and sensitivity. 42 Application of short PCR primers in RPA has been demonstrated by Mayboroda et al, 43 Martorell et al, 37 Wang et al 44 and Fuller et al 45 The latter two authors have shown that the PCR primers used in RPA resulted higher analytical sensitivity of detection compared to their usage in PCR: RPA detected 100 DNA copies of genetically modified GTS 40-3-2 soybean and 3.5 pg of genomic DNA of Agrobacterium spp. respectively, while the benchmark method PCR detected 1000 DNA copies and 350 pg of genomic DNA respectively.…”

“…proteinase K), protein sedimentation via high-speed centrifugation and purification using commercial DNA clean-up kit. 37,[109][110][111][112][113] Among these methods, the heating method worked equivalently to the methods by proteinase K digestion or SDS treatment. 109,111 However, heating at 65°C for 10 minutes showed better result than that of heating at higher temperature (95°C).…”

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

“…LAMP has been previously used to detect mutations in tumour tissue such as for KRAS mutations in colorectal colon cancer 32 using LAMP in tandem with ligation substrates. Two studies specific to the detection of PIK3CA mutations published recently involved a colorimetric assay using strand displacement amplification 33 , and recombinase polymerase amplification (RPA), which is another isothermal amplification method 34 . These other studies highlight the demand for a cost-effective and rapid method for mutational tracking of DNA markers in cancer.…”