Fluorometric detection of single-nucleotide mutations using tandem gene amplification

Highlights

• A tandem gene amplification method for detection of single nucleotide mutation (SNM) was developed.

• Circular padlock DNA specific to target SNM was used for generating G-quadruplex via Rolling Circle Amplification.

• Fluorescently visualized array-type multiple detection system was established for SNM detection in clinical samples.

• Mutant RNA was identified as low as 8.0 fg with a concentration as low as 0.53 % in the mixture with wild-type RNA.

Abstract

Sensitive and accurate identification of single-nucleotide mutations (SNMs) has become crucial in the field of molecular diagnosis and personalized medicine. Here, we developed a highly sensitive fluorometric assay for detecting multiple SNMs by tandem gene amplification; reverse transcription PCR (RT-PCR) coupled with rolling circle amplification generating G-quadruplex (GQ-RCA). In tandem gene amplification, RT-PCR-amplified DNA from sample RNA was digested with lambda exonuclease, which degraded the strand harboring the phosphorylated 5′-end of amplicon DNA to generate single-stranded DNA (ssDNA). The resulting ssDNA was hybridized with padlock probe DNA, which can discriminate a single base mismatch. Depending on the presence of mismatched bases between the amplicon ssDNA and padlock probe DNA, ligation of both ends of the padlock DNA was evaluated. If ligation occurred, a circular form of padlock DNA was generated, which was eligible for RCA. The RCA process generates long stretches of ssDNA containing tandem repeats of G-quadruplex structures. The amplified ssDNA harboring G-quadruplex was fluorescently visualized and quantified using Thioflavin T fluorophore. Our assay detected mutant RNA containing an SNM as low as 8.3 fg and detected mutant RNA with a concentration as low as 0.53 % in the mixture with wild-type RNA. The fluorometric SNM detection method with tandem gene amplification was successfully applied for multiplex detection of SNMs in clinical samples derived from patients with chronic myeloid leukemia. Our method would be useful in clinical diagnosis for early detection and monitoring of multiple SNMs in cancer patients with proper treatment.

Introduction

Over the past several decades, great advances in cancer genome analysis have revealed that tumorigenesis and tumor development are mostly associated with the accumulation of genetic mutations [1,2]. Identification of mutated genetic contents including single-nucleotide mutation (SNM) in oncogenes is essential for prognosis prediction and proper treatment of patients with cancer. Several SNMs in leukemogenic oncogenes can warn against treatment with tyrosine kinase inhibitors (TKIs). Among these, imatinib mesylate, a first-line treatment for breakpoint cluster region-Abelson (BCR-ABL) fusion gene-positive chronic myeloid leukemia (CML) [3,4], must be replaced with other regimens in cases with TKI-refractive SNMs. Various SNMs in the tyrosine kinase domain of BCR-ABL such as T315I point mutation were discovered in imatinib-resistant cases [[5], [6], [7]], which are known to inhibit imatinib binding to BCR-ABL chimeric protein by disrupting interactions between imatinib and the kinase domain of ABL or by inducing the activated conformation of BCR-ABL which is not suitable for imatinib binding [8,9]. Thus, sensitive and accurate detection of multiple SNMs related to TKI resistance is important for monitoring personal clinical information after treatment and making better decisions regarding cancer therapy.

Although sequencing-based technologies including Sanger DNA sequencing [10], next-generation sequencing [11], and nanopore sequencing [12,13] have been widely used to detect SNMs, they have technical limitations such as low sensitivity for detecting very small numbers of SNM genes in the abundant background of wild-type genes. Potential approaches for avoiding these challenges are to improve the SNM discrimination capacity by using several hybridization-based nucleic acid probes, such as allele-specific primers [14], double mismatched primers [15], double-stranded toehold exchange probes [16,17], transient binding short probe [18], zip nucleic acids [19], and peptide nucleic acids [20,21]. However, the discrimination capability of annealing-based methods should be enhanced to sensitively detect multiple SNMs. Alternatively, enzyme assays that can recognize sequence mismatches have been reported for SNM detection because of their simplicity and high specificity in the presence of high levels of wild-type genes [[22], [23], [24], [25]]. Among them, the ligase-mediated assay is considered as a versatile technology for detecting SNM genes with a high discrimination capacity [[26], [27], [28]]. For instance, in the presence of a corresponding target containing an SNM sequence, probe DNA can be ligated by enzymes such as Taq DNA ligase, which can in turn serve as templates for further amplification.

Rolling circle amplification (RCA) is an isothermal amplification method that can be combined with the ligation-mediated assay. After a ligated circular padlock template is obtained by an elaborate annealing process with a target SNM sequence in the ligation step, the long stretch of single-stranded DNA (ssDNA) product harboring the tandem sequence can be synthesized through the high productivity of phi29 DNA polymerase under isothermal conditions [29]. Several studies reported the RCA generating G-quadruplex system for the detection of target molecules including SNMs [[30], [31], [32]]. In this system, water-soluble thioflavin T (ThT) was used for fluorescence acquisition, which can selectively intercalate into G-quadruplex structures and emit strong fluorescence [[33], [34], [35]]. However, previous attempts to detect single base changes in the nucleic acid were confined to artificially prepared oligonucleotides of short lengths as sample molecules [30,36]. Clinical samples derived from patient tissues and blood plasma often contain trace amounts of double-stranded genomic DNA and long-length mRNAs with low abundance of the target gene. Thus, it is imperative to prepare suitable amounts of ssDNA for probe hybridization and subsequent enzyme assays [37,38]. To this end, preferential digestion of one strand in the PCR-amplified dsDNA amplicon has been developed by using lambda exonuclease and a 5′-end phosphorylated primer, which can produce large amounts of ssDNA of the intended length [[39], [40], [41], [42]].

In this study, we established a fluorometric system for highly selective and sensitive detection of SNMs present in the leukemogenic BCR/ABL fusion gene. Through tandem gene amplification by RT-PCR and subsequent G-quadruplex generating RCA, multiple drug-resistant SNMs in the ABL gene were fluorescently detected in an array type 96-well plate. This fluorometric system was applied to detect SNMs in sample ABL genes obtained from patients with CML, which provided 90 % matched detection results as compared to respective gene sequencing result. Our highly sensitive array-type fluorometric system will be useful for the early diagnosis of multiple SNMs in patients with CML, which will provide clinical information for proper treatment.