Showing papers on "Multiplex polymerase chain reaction published in 1993"

X-linked dilated cardiomyopathy. Molecular genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus.

Abstract: BACKGROUND X-linked cardiomyopathy (XLCM) is a rapidly progressive primary myocardial disorder presenting in teenage males as congestive heart failure. Manifesting female carriers have later onset (fifth decade) and slower progression. The purpose of this study was to localize the XLCM gene locus in two families using molecular genetic techniques. METHODS AND RESULTS Linkage analysis using 60 X-chromosome-specific DNA markers was performed in a previously reported large XLCM pedigree and a smaller new pedigree. Two-point and multipoint linkage was calculated using the LINKAGE computer program package. Deletion analysis included multiplex polymerase chain reaction (PCR). Dystrophin protein was evaluated by Western blotting with N-terminal and C-terminal dystrophin antibody. Linkage of XLCM to the centromeric portion of the dystrophin or Duchenne muscular dystrophy (DMD) locus at Xp21 was demonstrated with combined maximum logarithm of the scores of +4.33, theta = 0 with probe XJ1.1 (DXS206) using two-point linkage and +4.81 at XJ1.1 with multipoint linkage analysis. LOD scores calculated using other proximal DMD genomic and cDNA probes and polymerase chain reaction polymorphisms supported linkage. No deletions were observed. Abnormalities of cardiac dystrophin were shown by Western blotting with N-terminal dystrophin antibody, whereas skeletal muscle dystrophin was normal, suggesting primary involvement of the DMD gene with preferential involvement of cardiac muscle. CONCLUSIONS XLCM is due to an abnormality within the centromeric half of the dystrophin genomic region in heart. This abnormality could be due to 1) a point mutation in the 5' region of the DMD coding sequence preferentially affecting cardiac function, 2) a cardiac-specific promoter mutation that alters expression in this tissue, 3) splicing abnormalities, resulting in an abnormal cardiac protein, or 4) deletion mutations undetectable by Southern and multiplex polymerase chain reaction analysis.

Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs

Abstract: Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Abstract: Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.

PCR protocols: current methods and applications.

Abstract: Polymerase Chain Reaction. Selection of Primers for Polymerase Chain Reaction. Direct Radioactive Labeling of Polymerase Chain Reaction Products. Use of Arithmetic Polymerase Chain Reaction for Synthesis of Single-Stranded Probes for S1 Nuclease Assays. Nonradioactive Labeling of Polymerase Chain Reaction Products. Quantitation and Purification of Polymerase Chain Reaction Products by High-Performance Liquid Chromatography. Use of Polymerase Chain Reaction for Screening Transgenic Mice. Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. The Use of Polymerase Chain Reaction for Chromosome Assignment. Mapping MHC Class II Genes and Disease-Susceptibility. The Use of the Polymerase Chain Reaction and the Detection of Amplified Products. Determination of Loss of Heterozygosity Using Polymerase Chain Reaction. Direct Sequencing of Polymerase Chain Reaction Products. Manual and Automated Direct Sequencing of Product Generated by the Polymerase Chain Reaction. Genomic Footprinting by Ligation Mediated Polymerase Chain Reaction. RNA Template-Specific Polymerase Chain Reaction (RS-PCR). Quantitative Measurement of Relative Gene Expression in Human Tumors. Identification of Alternatively Spliced mRNAs and Localization of 5' Ends by Polymerase Chain Reaction Amplification. Utilization of Polymerase Chain Reaction for Clonal Analysis of Gene Expression. Sequencing DNA Amplified Directly from a Bacterial Colony. Use of Polymerase Chain Reaction to Screen Phage Libraries. Molecular Cloning of Polymerase Chain Reaction Fragments with Cohesive Ends. Rapid (Ligase-Free) Subcloning of Polymerase Chain Reaction Products. Use of Polymerase Chain Reaction for Making Recombinant Constructs. In Vitro Recombination and Mutagenesis of DNA. Use of Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes. Recombinant Circle Polymerase Chain Reaction for Site-Directed Mutagenesis. Site-Directed Mutagenesis by Double Polymerase Chain Reaction. Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA. PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy. Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members. Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking. cDNA Cloning by Inverse Polymerase Chain Reaction. Amplification of Gene Ends from Gene Libraries by Polymerase Chain Reaction with Single-Sided Specificity. Anchoring a Defined Sequence to the 5' Ends of mRNAs. Index.

Multiplex polymerase chain reaction for detection of genes for Staphylococcus aureus thermonuclease and methicillin resistance and correlation with oxacillin resistance.

Abstract: A multiplex polymerase chain reaction (mPCR) was used for simultaneous amplification of the staphylococcal nuc gene, encoding the thermostable nuclease (TNase), and the mecA gene, encoding the penicillin-binding protein 2a which is associated with staphylococcal methicillin resistance. A total of 219 staphylococcal strains were tested and the mPCR data were compared with coagulase production and in vitro oxacillin susceptibility. The agreement was 100% for coagulase production and nuc amplification, and 97.7%, 96.8 and 97.3% for mecA amplification and oxacillin resistance tested with MIC determination, disk diffusion and agar screen methods, respectively. Discrepant results were due to non-S. aureus isolates with borderline MICs of oxacillin (1-8 micrograms/ml). In a pilot test the mPCR simultaneously amplified both genes of staphylococci in blood cultures. This mPCR is a rapid and reliable method for single-step identification of cultures of MRSA and may prove to be useful for direct application on clinical specimens.

Selection of primers for polymerase chain reaction.

Abstract: One of the most important factors affecting the quality of polymerase chain reaction (PCR) is the choice of primers. Several rules should be observed when designing primers and, in general, the more DNA sequence information available, the better the chance of finding an "ideal" primer pair. Fortunately, not all primer selection criteria need be met in order to synthesize a clean, specific product, since the adjustment of PCR conditions (such as composition of the reaction mixture, temperature, and duration of PCR steps) may considerably improve the reaction specificity. Amplification of 200-400-bp DNA is the most efficient and, in these cases, one may design efficient primers simply by following a few simple rules described in this chapter. It is more difficult to choose primers for efficient amplification of longer DNA fragments, and use of an appropriate primer analysis software is worthwhile.

Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction

Abstract: We have optimized primers for cloning libraries of murine heavy and light chain variable regions using the polymerase chain reaction. Since we are interested in cloning murine Fab fragments for expression in bacterial cells, the heavy chain primers were designed to clone Fd fragments comprising the heavy chain variable domain and the first domain of the IgG constant region. The light chain primers were designed to clone the entire murine kappa chain. Using ten degenerate 5' primers and a degenerate 3' primer to amplify murine Fd and seven degenerate 5' primers with a single 3' primer to amplify kappa chains, a diverse repertoire of mouse variable regions was cloned from mouse spleens.

Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis.

Abstract: A rapid identification of Bacillus thuringiensis strains was established by using multiplex polymerase chain reaction (PCR). Primers of high homology specific to regions within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each strain of B. thuringiensis subsp. kurstaki. Differentiation among these strains was made on the basis of the electrophoretic pattern of the PCR products. Known B. thuringiensis subsp. kurstaki strains as well as unidentified strains isolated from insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a two-step PCR containing five primers capable of distinguishing between the strains giving products of 1,500, 858, and 653 bp for the CryIA(a) CryIA(b), and CryIA(c) genes, respectively. The method can be applied to rapidly detect the strains of B. thuringiensis subsp. kurstaki in commercial formulations and in the field.

Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method.

Abstract: We describe a two-step polymerase chain reaction method that can be used for the amplification of cellular DNA sequences adjacent to an integrated retroviral provirus. The technique involves a partly degenerate, arbitrary primer that will hybridize in the provirus-flanking cellular DNA. By using this primer in combination with a biotinylated provirus-specific primer, a provirus-cellular DNA junction fragment can be isolated from the nonspecific amplification products by using streptavidin-coated magnetic beads. A second amplification employing a nested provirus-specific primer and a biotinylated nondegenerate primer derived from the partly degenerate primer followed by purification with streptavidin-coated beads enhances the specificity and the efficiency of recovery of a fragment(s) containing the unknown flanking sequences. In addition to being relevant in studies of viral integration sites, the method should be generally useful to analyze DNA sequences either upstream or downstream from a known sequence.

Rapid identification of yeast species using three primers in a polymerase chain reaction.

Abstract: Classic methods for identification of yeasts rely on a variety of morphological and physiological tests that often take days to weeks to complete. We have been able to reduce the time to less than one day through the use of multiple segment-specific oligonucleotide priming of a region of the large subunit rDNA in a polymerase chain reaction. The "hot start" reaction was used with two universal external delimiting primers and one internal species-specific primer. Five specific primers were tested: a primer for a biologically similar group of Rhodotorula species, a generic (Cystofilobasidium) primer, and 3 species-specific primers (Leucosporidium scottii, Cryptococcus muscorum, and Rhodotorula mucilaginosa). In the absence of specific target DNA, the universal rDNA segment is amplified; in the presence of target DNA, the specific primer region is amplified. The technique is accurate within two base position differences when a 24 nucleotide-specific primer is used. The technique should be applicable to other marine eukaryotes.

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction.

Abstract: Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses. For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses. Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested. In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains. By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates. By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung. Such extracts were used directly for RT-PCR without prior RNA isolation. We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction. This allowed the differentiation of HCV from BVDV and BDV in one step. To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining. The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses.

Use of polymerase chain reaction to amplify segments outside boundaries of known sequences.

Abstract: Publisher Summary This chapter reviews the procedures adopted by several investigators to obtain fragments outside a region of known sequence and provide a protocol for one technique––inverse polymerase chain reaction (PCR)––used by the laboratory. Prior to the implementation of the PCR, the acquisition of specific DNA fragments usually entailed the construction and screening of DNA libraries, and the traditional approach for walking from regions of known sequence into flanking DNA involved the successive probing of libraries with clones obtained from prior screenings. Nevertheless, several applications, including the recovery of the flanking sequence and the generation of end-specific hybridization probes for chromosome walking, can be greatly facilitated by the PCR. The specificity of the reaction is, in most cases, supplied by the unique primer designed to the core region. No single method can be recommended for every application because the source (DNA, RNA, or inferred from protein sequence) and size of the core region as well as the requirements for flanking information vary with each experiment.

Cycle sequencing with non-thermostable dna polymerases

Abstract: Method for performing a cycled primer extension reaction by contacting a template DNA with a primer in the presence of sufficient glycerol or ethylene glycol to lower the melting temperature of template DNA and primer hybrids below 70° C. and a DNA polymerase under conditions in which the DNA polymerase can cause primer extensions and is stable to the temperature at which the reaction mixture is heated to denature the primer extension product from the template nucleic acid; and a kit suitable for use in cycle primer extension reaction including the necessary primers, buffers and enzymes required for the procedure, and glycerol.

Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission.

Abstract: A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.

Fast DNA isolation from Histoplasma capsulatum: methodology for arbitrary primer polymerase chain reaction-based epidemiological and clinical studies.

Abstract: The arbitrary primer polymerase chain reaction (also called random amplified polymorphic DNA, or RAPD) is a DNA fingerprinting method that provides an efficient, sensitive way of discriminating between independent isolates of Histoplasma capsulatum, but its widespread application has been hampered by the arduous 2-day procedure traditionally used to extract DNA from H. capsulatum. We present here a quick (approximately 2-h) extraction method and show that the resultant DNA is suitable for sensitive and reproducible identification of individual strains of this pathogenic fungus.

Purification of forensic specimens for the polymerase chain reaction (PCR) analysis

Abstract: Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis. DNA extracted from putrefied tissue or bloodstains sometimes contained the copurified contaminant, that was identified as the porphyrin compound (hematin). When contaminated but less degraded DNA was analyzed by PCR, it was necessary to eliminate the impurity by anion exchange column chromatography or chelating resin preparation, and ultrafiltration using Centricon microconcentrators. When highly degraded DNA was analyzed, trace amounts of high molecular weight DNA was recovered by electroelution method, and then further purified by both column chromatography and ultrafiltration. From thus purified samples, the amelogenin gene for sex determination could be amplified by dual PCR technique.

DNA Fragment Length Polymorphism Analysis of Mycobacterium tuberculosis Isolates by Arbitrarily Primed Polymerase Chain Reaction

Abstract: Strain identification of Mycobacterium tuberculosis would prove whether transmission had occurred between individuals. A method to characterize strains of M. tuberculosis has been developed utilizing polymerase chain reaction (PCR). Purified chromosomal DNA of cultured clinical samples of M. tuberculosis were subjected to PCR using short (10-12 nucleotide) oligonucleotide primers. PCR products visualized after agarose gel electrophoresis and ethidium bromide staining demonstrated that different strains of M. tuberculosis give different banding patterns. This technique was used to confirm the relationship between cases of tuberculosis in several clusters, prove the lack of relationship between 2 isolates with the same antibiotic-resistance pattern, confirm a suspected mislabeling event, and suggest the source of infection in a case of tuberculous meningitis. This method is rapid and simple and does not require radioactive probes.

Use of polymerase chain reaction for the rapid construction of synthetic genes.

Abstract: Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a simple two-step PCR method for the rapid construction of synthetic genes (3). This method is based on early observations by Mullis et al. (4) in which multiple overlapping oligonucleotides could be used to generate synthetic DNA through several sequential rounds of Klenow-based PCR amplification. The method described in this chapter utilizes the thermostabile Taq polymerase and allows for the generation of synthetic genes in as little as 1 d. This method has proven useful in studies in which synthetic genes were constructed for the HIV-2 Rev protein (3,5) and the Wilms' tumor locus zinc finger protein (6). Furthermore, this method has been successfully employed in extensive mutagenesis of the HIV-1 rev response element (7).

A rapid and reliable PCR method for genotyping the ABO blood group.

Abstract: The ABO blood group has been used extensively as a marker in population studies, epidemiology, and forensic work. However, until the cloning of the gene, it was not possible to determine the genotype of group A and B individuals without recourse to family studies. We have developed a method to determine the ABO genotype directly from human DNA using multiplex PCR and restriction enzyme analysis. Two PCR fragments spanning positions 258 and 700 of the cDNA sequence are amplified. The site at position 258 allows us to differentiate the O allele from the A and B alleles. The site at position 700 allows us to distinguish the B allele from the A and O alleles. Analysis at the two sites thus allows us to distinguish the three alleles. The multiplex PCR product is digested separately with four enzymes, two for each of the sites. The pair of enzymes for each site cut in a reciprocal fashion. Whereas one enzyme for each site is theoretically sufficient for genotyping, the use of complementary pairs of enzymes prevents the assignment of a false genotype as a result of false negative or partial digestion. This method is fast and reliable, does not rely on probing of blots, and should be widely applicable.