Showing papers on "Multiplex polymerase chain reaction published in 1992"

Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction

Abstract: Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

Polymerase Chain Reaction (PCR) Primers for Amplifying and Sequencing Nuclear 18S rDNA from Lichenized Fungi

Abstract: (1992). Polymerase Chain Reaction (PCR) Primers for Amplifying and Sequencing Nuclear 18S rDNA from Lichenized Fungi. Mycologia: Vol. 84, No. 4, pp. 589-592.

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

Abstract: A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.

Molecular Methods for Virus Detection

Abstract: D.L. Wiedbrauk and A.M. Drevon, Nucleic Acid Detection Methods. D.L. Wiedbrauk and J. Stoerker, Quality Assurance in the Molecular Biology Laboratory. D.L. Stoler and N.L. Michael, Nucleic Acid Blotting Techniques for Virus Detection. J.Carr, In Situ Hybridization. R.L. Hodinka, Antiviral Susceptibility Testing Using DNA-DNA Hybridization. J.C. Wilbur and M.S. Urdea, Quantitation of Viral Nucleic Acids Using Branched DNA (bDNA) Signal Amplification. I. Bronstein and C.E.M. Olesen, Chemiluminescence Methods. B.J. McCreedy, Polymerase Chain Reaction. F. Ferre, P. Pezzoli, E. Buxton, C. Duffy, A. Marchese, and A. Diagle, Quantitation of RNA Targets Using the Polymerase Chain Reaction. J.B. Mahony and M.A. Chernesky, Multiplex Polymerase Chain Reaction (M-PCR). G.J. Nuovo, PCR in Situ Hybridization. R. Sooknanan, B. van Gemen, and L.T. Malek, Nucleic Acid Sequence Based Amplification (NASBA(tm)). S.S.Ghosh, E. Fahy, and T.R. Gingeras, The Self-Sustained Sequence Replication (3SR) Reaction and its Application in Clinical Diagnostics and Molecular Biology. J.D. Burczak, S. Ching, H.-Y. Hu, and H.H. Lee, Ligase Chain Reaction (LCR) for the Detection of Infectious Agents. G. T. Walker, et al., A Chemiluminescent DNA Probe Test Based upon Strand Displacement Amplification (SDA). D.M. Schuster, M.S. Berninger, and A. Rashtchian, Ligation Activated Transcription Amplification: Amplification and Detection of Human Papillomaviruses. Subject Index.

Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.

Abstract: We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue ...

Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp

Abstract: Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.

Analysis of gene amplification in archival tissue by differential polymerase chain reaction.

Abstract: Oncogene amplification is found in many human tumors, and its detection may have important prognostic value. However, analysis of gene amplification may be hampered by inadequate tissue or poor DNA quality. We have previously described a polymerase chain reaction (PCR)-based procedure called differential PCR that can detect variations in gene dosage using miniscule amounts of tumor DNA [Frye, R.A., Benz, C.C. & Liu, E. (1989). Oncogene, 4, 1153-1157]. We now report the optimization of this technique for the analysis of oncogene amplification in paraffin-embedded archival tissues. We find that differential PCR is able to detect amplification of the HER2 (c-erbB-2) and the epidermal growth factor receptor (EGFR) genes and can be used to arrive at a semiquantitative estimate of gene dosage. Furthermore, our approach can determine gene amplification in samples in which the DNA is significantly degraded. Using differential PCR on paraffin-embedded tissues from cases previously investigated by standard DNA extraction and dot-blot procedures, good correlation between the two methods was found. Approaches are described to overcome technical problems posed by factors that affect the differential PCR, including the method of DNA extraction and extreme fragmentation of the DNA (less than 200 base pairs). Furthermore, the resulting analytical algorithm reported herein has proved effective in detecting oncogene amplification in archival breast cancer specimens from standard pathology laboratories. Thus, differential PCR will be particularly helpful in the analysis of tumor specimens that are archived, small in size or rare in occurrence.

Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene.

Abstract: A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.

Polymerase Chain Reaction (PCR) Amplification and Human Leukocyte Antigen (HLA)-DQα Oligonucleotide Typing on Biological Evidence Samples: Casework Experience

Abstract: The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed

Dna cycle sequencing

Abstract: Method for sequencing DNA which includes the following steps: providing a polynucleotide primer complementary to a region of the DNA, providing the DNA to be sequenced, and contacting that primer and DNA together in the presence of a DNA polymerase and between 1 and 3 dNTPs, at least one of the dNTP being labeled. The primer and DNA are contacted under conditions which allow extension of the primer by addition of one or more of the dNTPs to the primer to form an extended primer. The primer and DNA are then dissociated, generally be heating, and the contacting and dissociating steps repeated a plurality of times (usually 10-200 times). Finally, the extended primer is contacted with the DNA in the presence of a DNA polymerase (which is generally the same polymerase as used in the initial labeling step) all four dNTPs and a chain terminating agent.

The polymerase chain reaction. History, methods, and applications.

Abstract: The polymerase chain reaction (PCR) uses in vitro enzymatic synthesis to amplify specific DNA sequences. PCR amplification can produce approximately 100 billion copies of one molecule of DNA in a few hours. PCR has revolutionized research in the biological sciences and medicine, and has influenced criminology and law. Several major scientific discoveries, including purification of DNA polymerase and elucidation of the mechanism of DNA replication, were essential for development of the present PCR technology. An overview of these discoveries and early work on in vitro DNA synthesis are presented. Basic PCR methodology, instrumentation, advanced PCR techniques, and applications are also discussed in this review. Several new amplification systems are mentioned. PCR is an extremely important and simple technology for research and diagnostic analyses of DNA and RNA. PCR technology and other amplification procedures will continue to produce novel applications in basic research and clinical medicine.

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

Abstract: A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR) Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR) The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR) The concentration of the single primer is approximately 04 microM Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA

Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

Abstract: We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.

Rapid assays for amplification products

Abstract: A method of detecting a nucleic acid sequence of interest in the amplification product of a polymerase chain reaction or other primer directed reaction comprising the steps of: (a) incorporating in a polymerase chain reaction mixture or other primer directed reaction mixture at least one nucleic acid sequence complementary to said nucleic acid sequence of interest labeled (i) at the 3' end thereof, or (ii) at the 3' and the 5' end thereof with a compound capable of electrochemiluminescence; (b) conducting a polymerase chain reaction or other primer directed reaction; and (c) measuring the electrochemiluminescence of labeled amplification product.

Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification.

Abstract: Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups. These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis. This polymerase chain reaction test is a facile method for rapidly classifying P. solanacearum strains. Images

Utility of an internal control for the polymerase chain reaction

Abstract: The polymerase chain reaction (PCR) was used to amplify a 209 base-pair fragment of Mycoplasma pneumoniae DNA. The amplicon was transferred into a plasmid and a 680 base-pair piece of foreign DNA was inserted between the two amplimer sites. Plasmid DNA was added to the reaction mixture as an internal control for the polymerase chain reaction. Since the original hybridization target sites were included in this construction, one pair of amplimers could be used to amplify both the target DNA and the internal control DNA. Separation of internal control from target DNA after amplification was easily obtained on agarose gel electrophoresis. For the analysis of clinical samples with the polymerase chain reaction, the addition of internal control DNA allowed monitoring of the overall effectiveness of the amplification in each tube. With this technique approximately one-third of the tests were shown to be unsatisfactory due to technical errors or contaminating inhibitors. Adequate internal controls are necessary to avoid false-negative results with the polymerase chain reaction.

Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study.

Abstract: OBJECTIVE--To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR). DESIGN--DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive. SETTING--Fourteen university-affiliated and private genetic disease diagnostic laboratories. PATIENTS--Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene. MAIN OUTCOME MEASURES--The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored. RESULTS--The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%. CONCLUSIONS--Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.

Genetic analysis using the polymerase chain reaction.

Abstract: The ability of PCR to amplify a specific DNA segment from a complex template in an automated reaction has made molecular analysis more accessible both to basic research and diagnostic laboratories. Modifications of the basic PCR method using generic primers now allow DNA amplification even in the absence of specific nucleotide sequence information. Although PCR has proved extremely valuable for detecting the presence or absence of a given sequence (e.g. diagnosis of infectious disease pathogens like HIV), it is uniquely suited to analyze genetic variation in the amplified DNA. The capacity to analyze DNA variation from limited samples like hair or cheek scrapings has made genetic diagnosis and population genetics studies much simpler and the amplification of specific sequences from individual cells (e.g. sperm) has opened new approaches to genetic mapping.

Polymerase chain reaction-based methods for the detection of human papillomavirus DNA.

Abstract: The unprecedented sensitivity of the polymerase chain reaction (PCR) DNA amplification technology provides numerous advantages for the detection of human papillomavirus (HPV) DNA. In this chapter, the principles of the method and its advantages are discussed; disadvantages of the technology and proposed solutions are also presented. To accommodate the complexity of the many distinct HPV types, both type-specific and consensus PCR-based detection methods have evolved. Consensus primer systems, which afford the amplification of a broad spectrum of HPV types, have proven particularly useful in epidemiological studies and in novel virus identification. Since the introduction of PCR into the HPV field, there have been rapid advances and improvements PCR technology. However, standardization of PCR methods for HPV detection is essential for research to fully benefit from the technology. To that end, specific guidelines regarding experimental controls, assessment of analytic sensitivity and data interpretation are outlined here.

Characterization of a DNA probe for detection of Mycobacterium tuberculosis complex in clinical samples by polymerase chain reaction.

Abstract: We cloned and sequenced a DNA fragment from Mycobacterium tuberculosis for use in the identification of members of the M. tuberculosis complex. The DNA probe for culture confirmation had a sensitivity and a specificity of 100%. By using primers developed from this probe, the polymerase chain reaction detected 20 mycobacteria by ethidium bromide staining. This polymerase chain reaction system demonstrated 98% sensitivity and 100% specificity for detection of the M. tuberculosis complex in 200 sputum specimens.

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis.

Abstract: Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR.

Abstract: About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.

Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence.

Abstract: Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg2+, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.

Use of polymerase chain reaction to detect pathogenic strains of Agrobacterium

Abstract: The polymerase chain reaction (PCR), a sensitive tool for the identificationof specific regions of DNA present in small quantities, was used to detect the presence of T-DNA in 34 Agrobacterium strains from Vitis spp. Oligonucleotide primers, homologous to T-DNA regions from a wide and a narrow host range strain of Agrobacterium tumefaciens, were used to amplify portions of the T-DNA. In most cases the PCR results confirmed pathogenicity tests using detached leaves from Agrobacterium-free muscadine plants and DNA slot blot hybridization (...)