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.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.     

Differential pre‐amplification of STR loci for fragmented forensic DNA profiling

DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre‐amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre‐amplification strategy. In addition, pre‐amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre‐amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples.     

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.     

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification （RCA） technique. In the presence of DNA adenine methylation （Dam） MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons （detection probes）, resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.     

A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid

An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a “caged” inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.     

The development of miniSTRs as a method for high-speed direct PCR

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.     

DNA profiling of shahtoosh

The population of the Tibetan Antelope (Pantholops hodgsonii) has recently declined dramatically due to the illegal trade in its wool. The animal lives at high altitude and is protected from the extremely cold climate due to a coat of very fine wool. These hairs are highly sought for weaving a shawl called shahtoosh. The large-scale poaching of the antelope has resulted in the species being placed on CITES Appendix I. We report on a method of DNA profiling on a confiscated shahtoosh using the cytochrome b (cyt b) gene to produce a test that will identify unambiguously the presence of P. hodgsonii. Two shahtoosh samples were provided by the Council of Agriculture, Taiwan, and ten shawl samples of sheep wool (Ovis aries), cashmere from the Kashmir goat (Capra hircus), and pashmina from the Himalayan goat (C. hircus) were collected from local stores for comparison. Two primer pairs were used to amplify a 271 bp fragment of cyt b gene which would distinguish these three species. The resulting amplification products were directly sequenced. When the DNA sequences were compared with the sequences registered in GenBank and EMBL databases, the sequences with the highest homology were the cyt b genes of P. hodgsonii, C. hircus, and O. aries. This study demonstrates that there is still sufficient DNA present in the finished wool of a shahtoosh shawl to allow DNA typing and the method established was highly plausible to identify the CITES protected species.     

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.     

Universal fluorescent labeling of amplification products using locked nucleic acids

Amplification/hybridization‐based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two‐step amplification process that uses locus‐specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(‐47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(‐21) sequences at lower Tm values). Moreover, AT‐rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC‐rich substitutions. GC‐rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA‐containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.     

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.     

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser‐induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high‐throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue

Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.     

Ultrasensitive Electrochemiluminescence Biosensor for mRNA Based on Polymerase Assisted Signal Amplification

A novel biosensor for ultra‐trace mRNA sensing was constructed based on isothermal circular strand‐replacement polymerization (CSRP) to amplify the electrochmeiluminescence (ECL) signal by combining quantum dots (CdTe) as luminophore. After the hairpin‐like capture DNA was opened by hybridization with target mRNA, the additive primer (DNA1) was able to get access to its complementary sequence which is partially belong to the stem part and triggered a polymerization of DNA strand, leading to the release of target mRNA and another polymerization cycle. The remaining sequence of the stem part continued to hybridize with QDs labeled DNA, accomplishing ECL signal amplification. Target mRNA could be specifically assayed with a linear relationship between the signal intensity and the logarithm of concentrations of target DNA in the range of 1.0×10⁻¹⁴∼5.0×10⁻¹⁰ M, with a low detection limit of 1.4×10⁻¹⁵ M. The signal could discriminate perfect matched target mRNA from 1‐base mismatch sequence. This proposed ECL biosensor exhibited an efficient performance in serum sample, opening new opportunities for genetic target analysis in diagnostic and clinic biomedical fields.     

Electrophoretic detection of genetic variability among Schistosoma japonicum isolates by sequence-related amplified polymorphism

In the present study, sequence‐related amplification polymorphism (SRAP) was utilized to study the genetic variability among Schistosoma japonicum isolates from different provinces in China, using Schistosoma mansoni from Puerto Rico for comparison. Five out of ten tested SRAP primer combinations displayed significant polymorphisms among S. japonicum isolates from China, namely ME2/EM1, ME4/EM1, ME4/EM6, ME5/EM4 and ME5/EM5. Analysis of the 61 S. japonicum samples from China with five SRAP primer combinations identified a total of 83 reproducible polymorphic fragments. The number of fragments using each primer combination ranged from 14 to 19, with an average of 16 polymorphic bands per primer pair, and the size of fragment ranged approximately from 100 to 1000 bp. Representative‐specific SRAP fragments were excised from the gels, and confirmed by PCR amplification of genomic DNA using primers designed and based on the sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram was constructed. UPGMA clustering algorithm categorized S. japonicum isolates from China into nine clades and two lineages (representing the mountainous and lake/marshland regions). These results indicate the usefulness of the SRAP technique for revealing genetic variability among S. japonicum isolates from China, and the SRAP technique should be applicable to other living organisms.     

Sensitive detection of soy (Glycine max) by real-time polymerase chain reaction targeting the mitochondrial atpA gene

Detection of trace amounts of allergens is essential for correct labeling of food products by the food industry. PCR-based detection methods currently used for this purpose are targeting sequences of DNA present in the cell nucleus. In addition to nuclear DNA, a substantial amount of mitochondrial DNA (mtDNA) copies are present in the cytoplasm of eukaryotic cells. The nuclear DNA usually consists of a set of DNA molecules present in two copies per cell, whereas mitochondrial DNA is present in a few hundred copies per cell. Thus, an increase in sensitivity can be expected when mtDNA is used as the target. In this study, we present a reporter probe-based real-time PCR method amplifying the mitochondrial gene of the alpha chain of adenosine triphosphate synthetase from soy. Increase in sensitivity was examined by determining the minimal amount of soy DNA detectable by mtDNA and nuclear DNA (nDNA) amplification. Additionally, the LOD of soy in a food matrix was determined for mtDNA amplification and compared to the LOD determined by nDNA amplification. As food matrix, a model spice spiked with soy flour was used. Sensitivity of PCR-based soy detection can be increased by using mtDNA as the target.