Fluorescence coupled in-capillary DNA hybridization assay and its application to identification of DNA point mutation

Highlights

• We examined the ssDNA hybridization inside a capillary.

• We prove CE-FL to be a powerful method to resolve DNA hybridization.

• We achieve chromatographic separation of DNA.

• We discovered a novel strategy for the online detection of base mutation(s).

• The in-capillary assay of integrating multi-steps has been developed.

Abstract

We have developed a fluorescence coupled in-capillary DNA hybridization assay and applied it to the detection of DNA base pair mutation(s). A 15-mer thrombin-binding single-stranded DNA oligonucleotide labeled with 6-carboxyfluorescein (DNA1-FAM) was mixed and hybridized with its complementary DNA (cDNA1) via the in-capillary approach. A fluorescence coupled capillary electrophoresis (CE-FL) was used to measure the status and reaction rate of DNA double strands hybridization under different reaction conditions, and the results were recorded on the electrophoretograms. Recorded electrophoretograms have shown that the in-capillary assay has different DNA hybridization patterns compared to the post-capillary assay. The DNA hybridization efficiency of the in-capillary assay was affected by the molar ratio, the time interval of injection and the volume of DNA oligos. Interestingly, this assay could be used to detect one or two base pair mutation(s) in the target DNA. The in-capillary assay, characterized by an easy operation, reproducible results, and a low instrumental requirement, can be coupled to an online base pair mutation-based detection system, which would facilitate the related small-scale research analysis at non-major research centers.

Introduction

Genomic mutations including base substitution/insertion or deletion/duplication of a DNA fragment are often caused by DNA damage or recombination. Base pair mutations in genomic DNA are frequent events, which often lead to the alterations in biological traits. Detection of DNA base pair mutation is required for screening in congenital diseases [1] and the diagnoses of genetic diseases such as cancers [2], [3], [4]. A number of methods have been implemented for biomolecules interaction, such as surface Plasmon resonance (SPR) [5], [6], isothermal calorimetry [7], [8], high performance size exclusion chromatography (HPSEC) [9], [10], microarray [11], [12], [13], etc. Conventional methods used for the detection of base pair mutation(s) usually rely on agarose gel, polyacrylamide gel electrophoresis or PCR, which are time-consuming, labor intensive, and difficult to be coupled to the automated DNA mutation analyses, considering the facts that it usually consumes large amount of reagents and it takes more time in preparing the agarose gel and polyacrylamide electrophoresis gel, and low resolution results derived from both methods. PCR procedure usually takes multiple steps and it is not easy to couple with real time the online detection needs. Recently, several approaches have been developed for the accurate validation on the DNA base pair mutation(s), including oligonucleotide ligation [14], [15], [16], [17], [18], primer extension, allele-specific DNA hybridization [19], [20], [21], [22], [23], and electrochemical typing [24], [25]. However, these techniques heavily rely on the expensive instrumental investment and high maintenance cost including a specialized technical support personnel, which are usually a good fitting for a large-scale analysis available at top-level academic institutes instead of a small-scale analysis at non-academic centers/hospitals.

As a micro-scale technology with high separation efficiency, capillary electrophoresis (CE) has been successfully applied to the separation of biomolecules [26], [27], [28] such as polysaccharide, protein, nucleic acid and base mutation [29], [30], [31], [32].

CE-based mutation detection technologies are well represented by single-stranded conformation polymorphism analysis (CE-SSCP) and restriction fragment length polymorphism analysis (CE-RFLP). Holmila et al. [33] have used CE-SSCP assay to analyze the TP53 gene point mutations in human lung cancer specimens, and have found that the sensitivity and accuracy of the assay are comparable with that being derived from DNA gel electrophoresis. Shin et al. [34] have established a bacterial pathogen detection system based on CE-SSCP technique, which could simultaneously detect 8 bacterial pathogens in clinical settings. In addition, Kuypers et al. [35] have used CE-RFLP method to identify the ectopic DNA of chromosome 14 and 18 for the diagnosis of lymphoma. On the same line, Mitchell et al. [36] also used CE-RFLP approach to detect the allelic gene mutation of K-ras gene. Their results showed that CE could separate and quantitate BstNI fragments containing K-ras codon 12 mutation. Recently, our group has also developed a novel CE-assisted QDs and Molecular beacon (QD-MB) biosensor for the simultaneous detection of dual single-base pair DNA mutations [37]. However, the rapid and real-time detection system on DNA base pair mutation(s) is still facing a great challenge. It is highly demanded to develope such an online assay.

Herein, we have set up a simple and rapid method for the online detection of DNA hybridization and base pair mutations. Briefly, a 15-mer DNA oligonucleotide labeled with 6-carboxyfluorescein (named DNA1-FAM) and its completely complementary (cDNA1) were injected into the capillary sequentially. The steps of mixing, reaction, separation and hybridization were performed within the capillary (in-capillary assay). The effects of the oligos’ molar ratio, injection time and interval time/volume on DNA hybridization were systematically examined and analyzed using fluorescence coupled capillary electrophoresis (CE-FL). Importantly, we have applied this in-capillary assay to detect DNA base pair mutation(s) by the observations of a DNA base pair mismatch peak on the recorded electrophoretograms. This novel rapid detection approach on the DNA base pair mismatch is an expansion of the DNA fluorescence probe labeling-based method that can be easily adopted to the clinical and research settings and facilitate biological analysis.