Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR.
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Base Sequence↗
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Results for “Multiplex PCR”
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[Genetic analysis of 32 microsatellite loci in 13 families of Wuzhishan pig by multiplex PCR and gene scanning technique].
Wuzhishan pig is one of the rare and endangered breeds in china. They have the following characteristics such as :light body weight and small size, early sexually maturity, high meat quality and genetic identification with 6 approximately 8 pares litter size,body weight of born 0.3 approximately 0.4 kg, 15 approximately 16 kg at 6 month old, 35 kg at 2 years old, and so on. They may be used for laboratory utilization, comparative studies on human medical model, embryonic engineering, nutrition metabolism, sensitivity test on virus and bacteria, skin brut and tranfer, removing lipid, teeth and mouth cavity diseases, studies on cardiovascular model and evaluation of new medicine products. The polymorphisms of 32 microsatellites in 13 families of Wuzhishan pig in Hainan were Analysed. Number of alleles in each family was counted, mean heterozygosity and polymorphism Information content(PIC) were calculated. The results showed that number of alleles was 13.66, mean heterozygosity was 0.559 while polymorphism information content was 0.731. This revealed that genetic diversity is abundant in Wuzhishan pig in Hainan. These results have instructional significance for preserving breeds, selection and breeding, development and utilization of Wuzhishan pig in Hainan.
Alleles↗
Turkish population data for 15 STR loci by multiplex PCR.
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Alleles↗
Multiplex PCR with confronting two-pair primers for CYP1A1 Ile462Val, GSTM1, GSTT1, and NQO1 C609T.
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an effective genotyping method for single nucleotide polymorphisms (SNPs) in aspects of reducing time and costs for analysis. So far we have established PCR-CTPP conditions for tens of SNPs, including a triplex genotyping (Kawase et al., 2003). In the present study we report a quadruplex PCR-CTPP to genotype simultaneously four functional polymorphisms of carcinogen-metabolizing enzymes, CYP1A1 Ile462Val, GSTM1 null, GSTT1 null and NQO1 C609T, which were reported that they have significant associations with smoking-related cancers. We applied this method for 475 health check-up examinees to demonstrate the performance. Among the subjects, the genotype frequency of CYP1A1 Ile462Val was 56.8% for Ile/Ile, 38.1% for Ile/Val and 5.1% for Val/Val. The null type frequencies of GSTM1 and GSTT1 were 52.8% and 49.9%, respectively. And the genotype frequency of NQO1 C609T was 41.9% for C/C, 41.3% for C/T and 16.8% for T/T. Their distributions were similar to those reported for Japanese by other studies. To the best of our awareness, this is the first paper that reports the success in quadruplex PCR-CTPP. The applied polymorphisms are useful ones, which would be adopted not only for research purposes, but also for risk assessment of individuals exposed to carcinogenic substances. This convenient genotyping would be applied for cancer prevention especially in Asian Pacific regions, where expensive genotyping methods are hardly available.
Cytochrome P-450 CYP1A1↗
[Tag primer-nested/ multiplex PCR for detection of Plasmodium falciparum and Plasmodium vivax].
OBJECTIVE: To establish a sensitive, simple to use and low noise nested/multiplex PCR for simultaneously detection of Plasmodium falciparum (Pf) and Plasmodium vivax (P.v). METHODS: The tag primer amplification technique, software Primer Premier 5.0, NCBI-BLAST web resources and the matrix test were used to optimize the nested/multiplex PCR for detection of P.f and P.v with filter paper blood samples taken from malaria patients diagnosed by microscopy, and the results of the optimized nested/multiplex PCR and microscopy were evaluated. RESULTS: The sensitivity of the optimized PCR, determined by the examination of imitative filter paper blood samples, was about 1-2 parasites / microl for P.f and 5-10 parasites / microl for P.v. Primer-dimer and other PCR noise were removed. When 71 field filter paper blood samples taken from microscopically diagnosed patients (24 P.f, 47 P.v) were examined, the concordance between the optimized PCR and microscopy was 875% for Pf and 100% for P.v. CONCLUSION: The nested/multiplex PCR optimized by tag primer amplification technique is simple, with low noise and being able to detect Pf and P.v simultaneously. It is more sensitive in detecting cases with low parasitaemia and more accurate in identifying Plasmodium species than microscopy.
Animals↗
[The study on the technique of sexing bovine pre-implantation embryos with two-temperature gradient multiplex PCR].
For the stable, reliable, fast method of sexing bovine pre-implantation embryos is still play very important role in husbandry production, the amplification experiment under the condition of the two-temperature gradient PCR was done with bovine samples such as genome, cloning embryos, embryos respectively. As a result, the stable, simple, fast method of two-temperature gradient PCR for sexing bovine pre-implantation embryos was obtained, which only took 57 minutes to identify the embryo sex. A total of 30 dairy embryos were sexed with the two-temperature gradient PCR method in the study. 15 identified embryos (11 female, 4 male) were transferred into 15 recipient cows, among them 7 were pregnant after 60 days. In the end, 5 female calves were aborted in late pregnancy, and 1 female and 1 male dairy calf were born. The sexes of aborted and born calves were fully in accordance with the embryo sex predetermination with PCR method.
Animals↗
The use of a new multiplex PCR assay significantly improves the rapid detection of deletions in the dystrophin gene.
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Chromosome Deletion↗
[Detection of deletions by the amplification of exons (multiplex PCR) in Duchenne muscular dystrophy].
The polymerase chain reaction is a new powerful method for in vitro cloning of specific regions of DNA. The use of the heat-stable DNA polymerase made the reaction amenable to automation. This method greatly facilitates the detection of mutations which are responsible for Duchenne muscular dystrophy, via DNA amplification of multiple deletions prone exons from the DMD gene.
Chromosome Deletion↗
Detection of four different 11q23 chromosomal abnormalities by multiplex-PCR and fluorescence-based automatic DNA-fragment analysis.
A nested polymerase chain reaction (PCR) protocol was developed for rapid detection of four different 11q23 abnormalities by a single PCR assay. During each of the two PCR rounds a sense primer located within exon 5 of the MLL gene at 11q23 was combined with four different antisense primers, each located within possible translocation partner genes at chromosomes 4, 6, 9, and 19, respectively. Except for the MLL primer all primers used during the second round of nested-PCR carried a characteristic fluorescence label at their 5'-end. Agarose gel analysis of the PCR products was sufficient to discriminate between the absence of any of the four MLL rearrangements and the presence of at least one of them. Discrimination of the four different MLL translocation partner genes was not possible by agarose gel analysis due to a molecular heterogeneity of the 11q23 breakpoints resulting in PCR products of variable size. For this reason, automatic fluorescence-based DNA-fragment analysis was used to exactly define the MLL translocation partner genes if a positive result had been obtained by agarose gel analysis. In patients with leukemia, this assay may enable a fast and highly sensitive detection of different 11q23 abnormalities, which usually correlate with poor clinical prognosis.
Base Sequence↗
Gender determination by multiplex PCR amplification of alphoid repeat sequences from single cells.
Alphoid repeat sequences on the X and Y chromosomes were successfully and consistently coamplified to determine the sex of single cells. Buccal cells served as a convenient model for single blastomeres obtained from preimplantation embryos through in vitro fertilization. The potential use of this technique for preimplantation genetic diagnosis is discussed.
Female↗
Identification and phylogenetic analysis of Lactobacillus using multiplex RAPD-PCR.
Multiplex RAPD-PCR was used to generate unique and identifying DNA profiles for isolates of the genus Lactobacillus. The method that was used was based on the combination of two 10-mer oligonucleotides in a single PCR. The generated RAPD profiles enabled discrimination of all lactobacillus strains that were used in this study. A dendrogram was generated from the RAPD profiles. The results of genetic relatedness obtained from the dendrogram were compared with the results obtained using carbohydrate fermentation profiles. Most of the gastrointestinal isolates studied could not be grouped using carbohydrate fermentation profiles. The RAPD profiles provided sufficient information to prepare a dendrogram of genetic relatedness. The gastrointestinal isolates were clustered together on the dendrogram. Furthermore an isolate originating from the stomach (strain ML004) was closely related to Lactobacillus fermentum. It was concluded that multiplex RAPD-PCR was useful for characterisation and inference of relatedness of Lactobacillus isolates.
Animals↗
Detection and quantification of transgenes in grains by multiplex and real-time PCR.
Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.
Amino Acid Sequence↗
Set of novel tools for PCR primer design.
We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.
Algorithms↗
Prenatal molecular diagnosis of Duchenne and Becker muscular dystrophy.
OBJECTIVE: Duchenne and Becker muscular dystrophy (DMD/BMD) is an X-linked lethal recessive disease caused by mutations in the dystrophy gene. There is no efficient treatment for this serious and disabling disease. We established a combination method to detect carriers and perform prenatal diagnosis. METHODS: In our study, from 1994 to 2005, using a different combination of 5 methods, including SRY gene amplification, multiplex PCR, multiplex Fluorescence PCR capillary electrophoresis, multiplex ligation-dependent probe amplification (MLPA) and linkage analysis of short tandem repeats (STR), 36 prenatal diagnosis were performed for pregnancies at risk of having a DMD/BMD baby through amniocentesis. RESULTS: Fourteen out of 21 male fetuses were found to be affected and respective pregnancies were terminated. A combined diagnostic rate of 83% was achieved for 30 cases with deletions, duplications, and non-deletion mutations after tested by more than one method. CONCLUSION: Using a combined method, we can diagnoses patients and carriers in DMD families, and perform prenatal diagnosis for the risk fetus. MLPA provides a simple, rapid and accurate method for deletions and duplications of all the 79 DMD exons. MLPA method for DMD diagnosis is the first report in our country.
Female↗
Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.
Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.
CRISPR/Cas diagnostics↗
Recent advances and future developments in PGD.
PGD has now been practised for a decade. The basic techniques currently used involve embryo biopsy, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). Recent advances in molecular diagnostic techniques have included the use of fluorescent PCR, multiplex PCR and whole genome amplification. For cytogenetic analysis, many centres are now using five or more chromosome probes to examine for chromosome abnormalities, sexing and aneuploidy. Future improvements in molecular diagnosis include the use of quantitative PCR, DNA fingerprinting and microarray technology. Developments in methods to analyse chromosomes from a single cell have included interphase chromosome conversion, which has already been clinically applied, and the use of comparative genomic hybridization, which is still being developed. These methods will hopefully enable more accurate and a greater number of diseases to be diagnosed at the single cell level.
Chromosome Aberrations↗
Technical report: Part 2. Basic requirements for designing optimal PCR primers.
Designing optimal polymerase chain reaction (PCR) primer sequences is one of the critical factors for successful PCR with sensitive, specific, and assay-to-assay reproducible results. In this review, all the requirements of PCR primer sequences are summarized, such as location, size of amplicon, length of primers, nucleotide composition, Tm, 3' terminal hybridization strength and frequency, hairpin formation energy, primer-to-primer interaction, specificity, and location of mismatches to sequences of cross-hybridization. The report also discusses how to explore these various types of information for more advanced PCR applications, which include nested PCR, multiplex PCR, competitive PCR, long PCR, point mutation detection, degenerate primers, and PCR cloning.
DNA Primers↗
A simple and affordable method for high-throughput DNA extraction from animal tissues for polymerase chain reaction.
We have developed a very simple and inexpensive method for high-throughput DNA extraction from animal tissues. The procedure contains three steps (digestion, heating, and centrifugation) and it is compatible with the 96-well plate format commonly used in polymerase chain reaction (PCR) amplifications. The duration for processing a plate is about 1.5 h; therefore, one researcher can isolate DNA from up to 1000 samples during a single workday. A small piece of tissue (ca. 10-20 mg) yields enough template for at least 50-70 PCR amplifications, as demonstrated by using the processed samples as templates successfully for long distance PCR, multiplex PCR, and randomly amplified polymorphic DNA (RAPD) assay. The application of our method is expected to facilitate studies that require high-throughput DNA isolation for PCR amplification, such as genotyping by microsatellites for mapping and genetic diversity studies, as well as mutant screening in zebrafish.
Animals↗