High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

Two-color allele-specific polymerase chain reaction (PCR-SSP) assay of the leptin receptor gene (Leprdb) for genotyping mouse diabetes mutation.

An allele specific polymerase chain reaction (PCR-SSP) assay for genotyping the mouse leptin receptor (Leprdb) mutation and its wild type (Lepr+) gene was developed using two different fluorescent dye-labeled primers. First, we determined the Leprdb and Lepr+ allele by PCR-SSP assay with usual dye-unlabeled primers. However this method requires two separate PCR reactions because the amplified products specific for each allele are almost the same size. We further developed a simple and reliable two-color PCR-SSP method that uses a color complementation strategy to distinguish the Leprdb and Lepr+ alleles. Leprdb/Leprdb, Leprdb/Lepr+ and Lepr+/Lepr+ of mice (5 each) were clearly genotyped by the two-color PCR-SSP. We also performed PCR-direct sequencing for the same samples and confirmed the accuracy of this method. This method makes it possible to reduce the number of PCR reactions because both alleles are amplified in the same reaction mixture.     

Rapid detection of hypoxanthine-guanine phosphoribosyltransferase on cellogel

Zusammenfassung 541 Serumproben von nichtverwandten Deutschen aus dem Kölner Raum wurden auf ihre Pt- und C3-Typen untersucht. Bei 537 Seren fand sich eine Überein-stimmung der Pt A-Typen mit den C3 F-Typen, der Pt AB-Typen mit den C3 FS-Typen und der Pt B-Typen mit C3 S-Typen. Die C3-Varianten F0.65S, F0.6S, F0.5S wurden als Pt AB bestimmt, die C3-Variante F0.55S wurde als Pt B bestimmt. Die Frage der Vergleichbarkeit der Systeme sowie die Untersuchungsmethoden werden diskutiert.

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Comparative studies on posttransferrin (Pt) and C3 in the polymorphism of the third component of human complement

Summary Sera of 541 unrelated Germans from the Cologne area were examined for their Pt and C3 types. There was correspondence between Pt A and C3 F, Pt AB and C3 FS, and Pt B and C3 S in 537 sera. C3 variants F0.65S, F0.6S, F0.5S were determined as Pt AB, C3 F0.55S was determined as Pt B. The question of possible identity and the methods used are discussed.

     

Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.     

Determination of the rdw rat genotype by polymerase chain reaction with allele-specific primer (PCR-ASP).

The rat carrying the rdw mutation (rdw rat) is a dwarf mutant with hypothyroidism that is caused by a single G to C transversion in the thyroglobulin gene. Therefore, the development of a simple method for molecular-based genotyping of this mutation has been problematic. We have developed a rapid and simple genotyping method that provides identification of both the rdw and wild-type allele. This polymerase chain reaction with an allele-specific primer (PCR-ASP) method amplifies only the specific allele, wild or mutant type, by using 3'-terminal mismatched primers that pair only with the respective alleles. This assay should be of value for rdw colony control and rapid discrimination of rdw/rdw, rdw/+ and +/+ rats.     

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3' end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.     

A Single multiplexed allele-specific polymerase chain reaction for simultaneous detection of alpha1-antitrypsin S and Z mutations

Alpha1-antitrypsin (AAT) deficiency is an inherited disorder that can cause lung disease in adults and liver disease in adults and children. The S and Z mutations are the two most common mutations found in the AAT-deficient patients. We have developed a simple multiplexed allele-specific-PCR to detect both the S and Z mutations and the corresponding wild-type alleles. Polymerase chain reaction (PCR) product could be resolved on an agarose gel or using any fluorescent gel detection system. We obtained accurate genotyping results for the four alleles; the S, Z, and their corresponding wild-type alleles for all investigated samples simultaneously. The approach described in this paper is rapid, cost effective, and reliable and can also be adaptable into any laboratory setting because of its simplicity.     

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction

We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.     

A label-free DNAzyme sensor for lead(II) detection by quantitative polymerase chain reaction

A label-free sensor was developed for sensitive detection of lead(II), combining high selectivity of a Pb²⁺-dependent DNAzyme with enormous signal amplification of quantitative polymerase chain reaction (QPCR). Specifically, a substrate strand was designed to have two primer-hybridization sequences at either terminus. The presence of lead ion (Pb²⁺) catalyzed cleavage of the substrate strands. This resulted in a concentration decrease of the substrate strand that could be detected by QPCR. Compared with existing DNAzyme-based protocols for Pb²⁺ assay, this strategy circumvented the use of various optical or electrical labels that might be difficult to be synthesized. Also, the incorporation of QPCR furnished our approach with high sensitivity and superb reproducibility. In addition, QPCR allowed an immediate quantification of the cleavage efficiency that could be useful for evaluation of the DNAzyme activity. The results obtained revealed that our approach exhibited a dynamic response toward Pb²⁺ within a three-decade concentration range from 10 nM to 5 μM with a detection limit of 1 nM. This approach also demonstrated good selectivity against other metal ions that commonly coexisted with Pb²⁺.     

Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples

A polymerase chain reaction (PCR) assay was evaluated for detection of Opisthorchis viverrini eggs in the stool specimens of light and heavily infected individuals in Khon Kaen province of Thailand. A total of 75 fecal specimens were analyzed by PCR following DNA extraction. All the microscopically positive samples were positive by PCR, while 23 of 30 (76.6%) microscopically negative samples were also PCR positive. The sensitivity of the assay was 5 eggs/g of stool. This method is potentially useful in the diagnosis of human opisthorchiasis in endemic areas for treatment and in epidemiological investigations.     

Directed termination of the polymerase chain reaction: kinetics and applications in mutation detection.

We describe a PCR-based method (DT-PCR) that integrates both DNA amplification and directed chain termination into a single-step process. This method exploits unbalanced nucleotide concentrations to induce the polymerase chain reaction to terminate at specific nucleotide sites, leading to the generation of two sets of nested termination fragments from genomic DNA. The kinetic mechanism underlying the termination process is outlined and the application of this method to the detection and characterization of mutations in fragments as long as 1 kb is described. The method is effective for the analysis of both haploid and diploid genomes, and not only allows the recognition of both indels (insertions and deletions) and nucleotide substitutions, but also enables the determination of their position in a single-step fashion.     

Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) has been used to detect Listeria monocytogenes in whole milk at a level of 0.1 cfu per 30 ml. This high degree of sensitivity has been achieved following enzymatic digestion, polysulphonone membrane filtration and amplification of a nucleotide sequence within the promoter region of hlyA. Key elements of the procedure are the absence of enrichment culture and a complete solubilization of the membrane filter, ensuring total nucleic acid recovery. The simplicity of the protocol coupled with high sample volumes and exquisite sensitivity extends the relevance of PCR within food and environmental microbiology.     

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

The mutation process of microsatellites during the polymerase chain reaction.

We build a mathematical model for the mutation process of microsatellites during polymerase chain reaction (PCR) using the theory of branching processes. Based on the model, we develop a method to estimate the mutation rate of microsatellites per PCR cycle and the probability of expansion by maximizing a quasi-likelihood of the observed data. We show by simulations that the proposed estimation method can accurately recover the relationship between the mutation rate and number of repeat units. The theoretical basis for the proposed method is also given. We apply the method to experimental data on poly-A and poly-CA repeats.