Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis
© Hur et al. 2015
Received: 18 September 2015
Accepted: 27 October 2015
Published: 4 November 2015
Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization.
Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe.
The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.
The introduction of advanced DNA sequencing technology has made a remarkable improvement in discovering the genetic variations or mutations in the human genome, virus, bacteria, and plants [1–3]. Detection of Single Nucleotide Polymorphisms (SNPs) and short insertions and deletions (indels) is a common goal in high-throughput sequencing experiments. Indel variations, the most common type of structural variance in the genome of an organism, affect a multitude of traits and diseases in the organism. Moreover, identification of genotypes in organisms using species- and genotype-specific DNA markers is very useful for species identification, diagnostics, and breeding and preservation programs. New sequencing technologies, such as deep sequencing, allow massive throughput of sequence data and greatly contribute to genetic variation studies.
Despite the advances of new sequencing technologies, we still face a limitation in the number of screening technologies, which include probe-based real-time PCR, specifically chip or sequencing [4–6]. Real-time PCR assays using fluorescence resonance energy transfer (FRET) probes, molecular beacons, or TaqMan probes have been adapted for continuous mutation detection of amplification products in a closed system. Nevertheless, these assays do not effectively distinguish the differences between wild type and mutant types of SNP(s), insertion(s), or deletion(s) because of several handicaps . First of all, traditional probe systems that have been used in SNP detection use two or more fluorescence channels for one mutation loci. When it comes to more than quadruplex detection, it can be a major problem in using real-time PCR. Moreover, traditional methods require ether the use of modification bases, special enzymes, or additional proprietary reagents or procedures. To solve these problems, we have adopted dual-labeled (fluorescence and quencher), full base peptide nucleic acid (PNA) hybridization probes for characterization of mutation assays [8, 9].
PNAs are artificially synthesized DNA analogs with an uncharged peptide backbone . They have more favorable hybridization properties and chemical, thermal, and biological stability because of their uncharged nature and their peptide bond-linked backbone . Because of these favorable characteristics, PNA probes are designed shorter (9–13 bp) than DNA probes with the same melting temperature (Tm). These characteristics make PNA probe more acceptable to use in mutation detection studies because PNA probe makes a large difference in ΔTm between perfect match and single mismatch, including even insertion and deletion. In this study, dual-labeled PNA probes were used to analyze genetic mutations including SNP, insertion, and deletion.
Description of dual-labeled PNA probe-based FMCA for SNP genotyping
Detection of insertion and deletion
Multiplex mutation detection in a short target region
Quantitative analysis of PNA probe-based FMCA
Adjustability of PNA probe hybridization temperature
Detection and discrimination between HRV and other rhabdovirus species with PNA–FMCA system in vitro
There are many ways to genotype nucleotide differences or changes in genome . Currently available techniques that require a separation step include restriction fragment length polymorphism analysis, single-nucleotide extension, oligonucleotide ligation, and direct sequencing. Additional methods, including pyrosequencing  and mass spectroscopy , are technically complex but can be automated for high-throughput analysis. Furthermore, real-time PCR-based assays that use high resolution melting (HRM) and probe-based systems, FRET probes, molecular beacons, or TaqMan probes have been adapted for continuous mutation detection of amplified products in a closed system. However, their capacity to discriminate DNA variants in the single nucleotide indel as well as SNP variant with single probe has not been examined until now. Furthermore, traditional probe systems that have been used in SNP detection use two or more fluorescence channels for one mutation loci. Thus even a wide range of mutation detection methodologies exist, DNA sequencing technology has been considered as the gold standard. However, several drawbacks have been reported in DNA sequencing, including its interpretation errors  and difficulty of high-throughput data generation, which makes DNA sequencing difficult to use. To solve these problems, we have adopted the full base PNA hybridization probes  for characterization of genetic variation assays.
The melting temperature (Tm) of nucleic acid oligomer is a physical parameter of nucleic acid hybrid. Under constant reaction conditions of heating rate, salt concentration, and probe-target concentrations, Tm is highly reproducible. Recently, HRM  and DNA, LNA probe melting analysis have been introduced and used for genotyping studies . However, multiplexing obtained by HRM alone will be limited and probe-based melting technologies face a difficulty of probe design, and low resolution (temperature difference) of variants remains to be limited by using existing probe chemistries (DNA, LNA. etc.).
To solve these problems, we adopted full base peptide nucleic acid (PNA) oligomer to probe-based melting point analysis. PNAs are artificially synthesized DNA analogs with an uncharged peptide backbone. PNAs have more favorable hybridization properties because of their uncharged nature and their peptide bond-linked backbone . Because of these favorable characteristics, PNA probes are able to be designed shorter (9–13 bp) than DNA probes (20 bp or more) with the same Tm. These characteristics make PNA probe more acceptable to use in genetic variation detection studies because PNA probe makes a large difference in ΔTm between perfect match and partial mismatch, including even insertion and deletion. In this study, PNA probes were used dual-labeled probe-based real-time PCR melting point anlysis to analyze genetic mutations include SNP, insertion, and deletion for genetic difference detection. The developments in self-quenching PNA real-time PCR probe have played a crucial role in the emergence of PNA probe-based melting analysis.
In here, we have shown that PNA probe-based melting system is a suitable method for nucleic acid change detection, as it was able to sensitively discriminate insertion(s), deletion(s), and SNP(s) along with the number of variations (Figs. 2 and 3). In case of narrowly distributed mutations (three mutations within 32 bp) (Fig. 4), HRM or DNA probe¬-based melting point assay may show erroneous results, because one amplicon or DNA probe contains multiple mutations that counteract each other, which causes differences in the Tm . In this study, the problem was completely resolved using dual-labeled PNA probes with two 5’-end quencher and 3’-end fluorescence conjugated PNA probe and one reversely conjugated PNA probe (Fig. 4a).
In addition to the genotyping studies, we have characterized PNA probe and asymmetric PCR conditions to verify their applicability. Genotyping analysis was measured without amplification measuring steps because of its time-consuming disadvantage. Therefore, to confirm the ability to measure the PCR amplification curve and DNA copies by dual-labeled PNA probe system, the target DNA was 10-fold diluted and amplified. Amplification curve (Fig. 5a) and standard curve (Fig. 5b) represent the possibility of quantitative analysis with dual-labeled PNA probe corresponding to its dilution ratios. This applicability was further confirmed by virus g-protein gene analogues detection study (Fig. 7). Furthermore, to detect amplification curve in the PNA-based probe system for quantitative analysis, the detection temperature depends on the primer annealing temperature. If probe-binding temperature is lower than that of the primer, the detection step will be added to detect fluorescence signal of lower Tm probe .
Moreover, shorter probe length with higher Tm was highly recommended due to the fact that shorter probe length makes ΔTm between perfect match and mismatch greater. Therefore, modifiability of probe Tm value plays a pivotal role in probe-based melting point analysis. We propose that there are two different ways to regulate Tm of a PNA probe. First of all, Like DNA oligomers, the Tm value of PNA probe radically depends on their length (Fig. 6a). Secondly, PNA oligomer is easily modifiable because of its peptide backbone . Gamma-modified PNA oligomer offers structural formation and ionic positive charges, which make PNA probe-binding efficiency higher . In this study, the Tm value of PNA probe was delicately affected by the number of gamma (Ala)-modified PNA oligomers of probe sequences (Fig. 6b). The PNA probes have varied ranges of Tm along with the number of gamma (Ala)-modified PNA oligomers (approximately 3–15 °C).
Here we show that peptide nucleic acid (PNA) is apt material for real-time PCR fluorescence probe for melting point analysis because it makes a large difference in the melting temperature (ΔTm) between full specific hybridization and that of single mismatch, including insertion and deletion. Furthermore, it is possible that PNA fluorescence probe effectively classify the co-existed SNP, insertion and deletion within short amplicon (32 bp) without any interference, which completely resolve the problem of high resolution melting (HRM) genotyping method. Moreover, applicability of PNA fluorescence probe is confirmed by the gamma modification of PNA that can simply substitute minor groove binder (MGB) of DNA probe with specific hybridization. Taken together, these characteristics demonstrated that dual-labeled and gamma-modified PNA probe greatly simplifies the probe design and dramatically increases allele-discriminating ability.
PNA oligomer and DNA target
All PNA probes (FAM-, HEX-, Texas Red-, or Cy5-labeled with quencher) were purified using high-performance liquid chromatography (Panagene, Korea), and the target oligonucleotides were synthesized and purified using polyacrylamide gel electrophoresis (Neoprobe, Korea). The purity of all the probes was confirmed by mass spectrometry. Unwanted secondary structure in the probe was avoided for better hybridization with its target. The mutation points were basically located in the center of the probe so as to obtain a Tm shift over 5 °C. For detection of deletion and insertion, end position deletion/insertion and center position deletion/insertion were used to optimize Tm shift. Single-stranded DNA oligomers were used as target DNA for probe validation, and each target DNA contained mismatch(es), deletion(s), or insertion(s). For mutation detection using PCR cyclic steps, synthetic double-stranded DNA containing two primers and probe-binding site(s) was used as the target. Four synthetic double-stranded DNA targets were randomly designed with 80 bp of DNA, containing one probe and two primer-binding sites, to analyze the ability to detect SNP. To analyze the differences in Tm between perfect match and deletion or insertion, three double-stranded DNA targets containing four perfectly matched probe-binding sites and four deletion or insertion sites in a single amplicon were artificially synthesized because of the cost efficiency in synthesizing double-stranded DNA. To analyze detection ability in a harsh condition, target DNA containing one SNP, deletion, and insertion in 118 bp DNA with a gap of 15 and 17 bp between each mutation point was synthesized. To avoid quenching of fluorescence signal within other probes, fluorescence and dabcyl (quencher) positions of Alexa488-probe were switched. The PNA oligomer, DNA target, and primer sequences are listed in Additional file 2: Table S1.
Direct analysis of PNA probe Tm value using fluorescence melting point analysis (FMCA) with synthetic oligonucleotides
Hybridization between probes and synthetic oligonucleotide targets was performed in a CFX96™ Real-Time System (BIO-RAD, USA). To analyze the Tm of PNA probe, synthetic single-stranded DNA oligomers were directly used as the target DNA. A thermal cyclic reaction was performed using the following conditions: the 20 μL reactions contained 1X SSB Real-Time FMCA™ buffer (SeaSun Biomaterials, Korea), 0.5 μM PNA probe, and 0.5 μL of DNA template (1 × 105 copies of synthetic DNA). Melting point analysis began with a denaturation step of 3 min at 95 °C; a stepwise hybridization and followed by a stepwise temperature increase up to 85 °C at 0.5 °C/step with a 5 s interval between each step (Additional file 4: Figure S1). The target oligomers and probes used are listed in Additional file 2: Table S1.
PNA probe-based FMCA for detection of genetic variation with PCR amplification
PCR and thermal cyclic reaction and hybridization between probes and synthetic oligonucleotide targets were performed in a CFX96™ Real-Time System (BIO-RAD, USA). In all the PCR amplification conditions, asymmetric PCR was used to generate single-stranded DNA target. An asymmetric PCR was carried out using the following conditions: the 20 μL reactions contained 1X SSB Real-Time FMCA™ buffer (SeaSun Biomaterials, Korea), 2.5 mM MgCl2, 200 μM dNTPs, 1.0 U Taq polymerase, 0.05 μM forward primer and 0.5 μM reverse primer, 0.5 μM PNA probe, and 0.5 μL of DNA template (1 × 105 copies of synthetic DNA). Real-time PCR and FMCA protocols started with a denaturation step of 7 min at 95 °C, followed by 32 ~ 50 cycles of 95 °C for 10 s, 55 °C for 15 s, and 74 °C for 30 s. Melting point analysis began with a denaturation step of 3 min at 95 °C, hybridization step, and followed by a stepwise temperature increase from 25, 30, or 35 °C to 85 °C at 1 °C/step with a 5 s interval between each step (Additional file 4: Figure S1). The forward and reverse primers and probes used are listed in Additional file 2: Table S1.
Hetero-type variation detection with PNA-based FMCA
To analyze hetero-type variation detection, DNA template consists of mutation type SNP was mixed with DNA does not consist of the SNP, with varied percentages (from 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to 100 %) and used as a template. Mutant-type target DNA was serially diluted in wild-type target DNA. Both perfect match and mutation templates were artificially synthesized and start copies were roughly 2 × 105 copies per reaction. The PNA-based FMCA was performed according to the conditions mentioned above.
Multiple mutation detection in a short target region
Multiple mutation detection was performed using the following conditions: the 20 μL reactions contained 1X SSB Real-Time FMCA™ buffer, 2.5 mM MgCl2, 200 μM dNTPs, 1.0 U Taq polymerase, 0.05 μM forward primer and 0.5 μM reverse primer, 0.5 μM probe per each fluorescence (Alexa488, HEX, Texas-red), and 0.5 μL of DNA template (1 × 105 copies of synthetic DNA). The same real-time PCR and FMCA protocols mentioned above were used.
Quantitative analysis of PNA-based FMCA
Serially diluted DNA template (1 × 108 to 1 × 102 copies of synthetic DNA) was used for quantitative analysis of PNA-based FMCA. Real-time PCR and FMCA protocols were performed as per the conditions mentioned above.
Detection and discrimination between HRV and other rhabdovirus species with PNA–FMCA system in vitro
Self-quenching PNA probe was designed (Dabcyl-TCACTCAACTGGAG-Texas-Red) using specific region of the glycoprotein of HRV (CTCCAGTTGAGTGA), IHNV (CTCCAGTGGAGTGA), and VHSV (CTCCAATTGAATGA) sequences that was discriminated by perfect match (HRV), single mismatch (IHNV), or two mismatches (VHSV). Synthetic DNA (276 bp) targets were amplified using PCR and hybridized with the chosen PNA probe containing an SNP site(s) mid-sequence. The PNA-based FMCA was performed as per the conditions mentioned above. The sensitivity test of this procedure was conducted using 10-fold-diluted synthetic DNA targets (109 to 1 start copies).
This work was supported by a grant from the National Fisheries Research and Development Institute (NFRDI, R2015069) in Korea.
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