Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.     

Human Y-chromosome haplotyping by allele-specific polymerase chain reaction

We describe the application of allele-specific PCR (AS-PCR) for screening biallelic markers, including SNPs, within the nonrecombining region of the human Y-chromosome (NRY). The AS-PCR method is based on the concept that the perfectly annealed primer-template complex is more stable, and therefore, more efficiently amplified under the appropriate annealing temperature than the complex with a mismatched 3'-residue. Furthermore, a mismatched nucleotide at the primer's 3'-OH end provides for a poor extension substrate for Taq DNA polymerase, allowing for discrimination between the two alleles. This method has the dual advantage of amplification and detection of alleles in a single expeditious and inexpensive procedure. The amplification conditions of over 50 binary markers, mostly SNPs, that define the major Y-haplogroups as well as their derived lineages were optimized and are provided for the first time. In addition, artificial restriction sites were designed for those markers that are not selectively amplified by AS-PCR. Our results are consistent with allele designations derived from other techniques such as RFLP and direct sequencing of PCR products.     

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

Detection of terminal mismatches on DNA duplexes with fluorescent oligonucleotides

This paper describes the design of terminal-mismatch discriminating fluorescent oligonucleotides (TMDFOs). The method is based on the use of sets of oligo-2'-deoxyribonucleotide probes linked via their 5'-ends, and varying-sized flexible polymethylene chains, to thiazole orange, with the linker being attached to the benzothiazole moiety. The sequence of each set of labelled probes was identical and complementary to the sequence to be analyzed on the single-stranded nucleic acid target except at the interrogation position, located at the 5'-end of the probes in a position adjacent to the attachment site of the label, where each of the four nucleic bases were incorporated. This work allowed the selection of probes showing, upon their hybridization with the target sequence, good discrimination between the matched and the mismatched duplexes under non-stringent conditions, with the mismatched duplexes being more fluorescent than the perfectly matched ones.     

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.     

微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes

Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.     

Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele-specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium-bromide-labeled fragments.     

Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR(AS-PCR) approach was applied to investigating-55C/T polymorphism in promoter region of the uncoupling protein 3(UCP3)gene.AS-PCR is a competitive PCR method which is based on positioning the 3' base of a PCR primer to match one single nucleotide polymorphism(SNP) allele and accurately extend only the correctly matched primer.But it is limited in use because of its poor specificity.In this study,we improved the specificity of AS-PCR by introducing additional mismatch at the pe...     

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.     

Analysis of the factors affecting the accuracy of detection for single base alterations by oligonucleotide microarray

The oligonucleotide microarray, a high-throughput polymorphism detection technology, holds great promise for the characterization of complex genetic variance. To achieve greater sensitivity and specificity for it to be an effective platform technology we present results and discuss some of the factors influencing signal intensities and single-mismatch discrimination in array-based mutation/SNP detection. Probes with a series of concentrations were spotted onto the slide in order to find the optimal concentration with the identifiable satisfying signals and the stable ratios between matched and mismatched probes. It was found that under our experimental conditions, when the initial probe concentration is higher than the maximum immobilization capability of the slide (7.5 microM), the hybridization signal will be saturated and the ratio between matched and mismatched probes will be more stable than at a lower probe concentration. Considering the cost of probes and the systematic stability, a constant spotting concentration of 10 microM was selected. The stability of different types of mismatched oligo-DNA duplexes on the glass surface was also confirmed. The results show that the order of stability of mismatched oligo-DNA duplexes on a glass surface is in general agreement with previous reports conducted using liquid and polyacrylamide gel pads. This suggests that the influence of the mismatched base pair on the stability of the duplex in a solid hybridization system is similar to that in the solution hybridization environment.     

Increased single nucleotide discrimination in arrayed primer elongation by 4'C-modified primer probes

Herein we describe the beneficial impact of immobilized 4'C-modified primer probes on detecting single nucleotide variations in arrayed primer extension by a DNA polymerase.     

An electrochemiluminescent assay for high sensitive detection of mercury (II) based on isothermal rolling circular amplification

In this study, we firstly demonstrated that Bst DNA polymerase shows specific recognition and function on the T–Hg²⁺–T biomimetic structure. Based on this, a novel available electrochemiluminescence (ECL) sensor for Hg²⁺ has been developed. In this strategy, magnet beads tagged primer was designed to complementary to the region of the circular padlock probe but with two T–T mismatches at the 3′ end. The mismatched primers cannot be extended by Bst DNA polymerase in the absence of Hg²⁺. Stable T–Hg²⁺–T can be formed in the presence of Hg²⁺, thus induces the elongation and amplification reaction by DNA polymerase with a rolling circular amplification (RCA) mechanism. Subsequently, the resulted RCA products are hybridized with the tris (bipyridine) ruthenium (TBR)-tagged probes and detected by ECL platform. Current method shows a sub-nanomolar sensitivity and excellent selectivity over a spectrum of interference metal ions.     

Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100 mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.     

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains.     

An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR

Single-step aqueous cross-coupling reactions of nucleobase-halogenated 2'-deoxynucleosides (8-bromo-2'-deoxyadenosine, 7-iodo-7-deaza-2'-deoxyadenosine, or 5-iodo-2'-deoxy-uridine) or their 5'-triphosphates with 4-boronophenylalanine or 4-ethynylphenylalanine have been developed and used for efficient synthesis of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8-substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7-substituted 7-deazaadenine and 5-substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two-step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.