Showing papers on "Multiplex polymerase chain reaction published in 1994"

Multiplex PCR: advantages, development, and applications.

Abstract: Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 Ever since it was shown that PCR could simultaneously amplify multiple loci in the human dystrophin gene, (1) multiplex PCR has been firmly established as a general technique. A short list of multiplex PCR applications now includes pathogen identification, gender screening, linkage analysis, forensic studies, template quantitation, and genetic disease diagnosis. Multiplex PCR can be a two-amplicon system or it can amplify 13 or more separate regions of DNA. It may be the end point of analysis, or preliminary to further analyses such as sequencing or hybridization. The steps for developing a multiplex PCR and the benefits of having multiple fragments amplified simultaneously, however, are similar in each system. This review focuses on multiplex systems in which each primer pair targets a single locus, unlike RAPD (2) and alumorph {3) PCR reactions, which amplify multiple loci with a single primer or primer set.

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory.

Abstract: A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

An optimized multiplex polymerase chain reaction (PCR) for detection of BCR-ABL fusion mRNAs in haematological disorders.

Abstract: A rapid and simple polymerase chain reaction (PCR) method is described that is capable of identifying any of the BCR-ABL transcripts that have yet been described in chronic myeloid or acute lymphoblastic leukaemia. Randomly primed cDNA is synthesized from leucocyte RNA and amplified in a single reaction containing four oligonucleotide primers (multiplex PCR). Different size products are generated from ela2 (p190) and b3a2 or b2a2 (p210) BCR-ABL transcripts which are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. Chronic myeloid leukaemia cells are readily detectable even when diluted 1 in 1000 with normal blood. Samples which do not have BCR-ABL rearrangements produce a single band derived from the normal BCR gene, and the presence of this band controls for adequate RNA and cDNA preparation. Using this assay we have detected BCR-ABL transcripts in a variety of haematological disorders.

Surveys of gene families using polymerase chain reaction: PCR selection and PCR drift

Abstract: with the potential to be phylogenetically informative (e.g., sequence variation, vari? ation within gene clusters, and number of gene clusters [Koop et al., 1989; Bartels et al., 1993; Cartwright et al., 1993; Pendleton et al., 1993]). Nevertheless, this complexity is sometimes difficult to interpret in a phy? logenetic context (e.g., orthologous and paralogous variation and gene conversion [Fitch, 1970; Hillis and Dixon, 1991; San? derson and Doyle, 1992]). Analysis of the sometimes complex characteristics of mul? tigene families has recently been greatly facilitated by use of the polymerase chain reaction (PCR). Using degenerate primers corresponding to highly conserved regions of homologous genes, PCR can be used to detect and identify members of these gene families in samples of genomic DNA or cDNA. The utility of such an approach has recently been demonstrated using degen? erate primers to amplify a region of Antennapedia-class homeoboxes (Murtha et al., 1991; Pendleton et al., 1993). This tech? nique, however, cannot be fully exploited until certain methodological issues have

Homozygous Deletions of 9p21 in Primary Human Bladder Tumors Detected by Comparative Multiplex Polymerase Chain Reaction

Abstract: Deletion mapping studies of primary bladder tumors have identified nonoverlapping areas of loss on each arm of chromosome 9, indicating that two distinct tumor suppressor loci are located on this chromosome. The deleted region on the p arm overlaps an area of 9p previously reported to be lost in a variety of neoplasms. Detailed loss of heterozygosity analysis of 9p in 112 primary bladder tumors using 12 microsatellite markers identified a minimal area of loss around the alpha-interferon locus at 9p21-22. Frequent homozygous deletions of the alpha-interferon locus were then identified in these tumors by a novel, comparative, multiplex polymerase chain reaction assay and were subsequently confirmed by Southern analysis. Based on these deletions, a putative tumor suppressor gene locus involved in bladder tumorigenesis was localized to a 10 cM region (flanked by D9S162 and D9S171), previously implicated in the progression of many neoplasms. Application of the multiplex polymerase chain reaction-based assay will allow rapid identification of homozygous deletions in many neoplasms and thus aid in mapping studies of critical suppressor genes.

Frequency of allele loss of DCC, p53, RBI, WT1, NF1, NM23 and APC/MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction.

Abstract: We report here the use of multiplex fluorescent polymerase chain reaction (PCR) for quantitative allele loss detection using microsatellites with 2-5 base pair repeat motifs. Allele loss of APC, DCC, p53 and RB1 in colorectal tumours has been reported previously using a variety of methods. However, not all workers used intragenic markers. We have used microsatellite polymorphisms which map within, or are closely linked to, these tumour-suppressor gene loci in order to determine whether these loci are indeed the targets for alteration in colorectal cancer. In addition, we have assayed two other tumour-suppressor genes, WT1 and NF1, to see whether they play a role in colorectal carcinogenesis. The putative metastasis-suppressor gene, NM23, was also investigated since there have been conflicting reports about its involvement in colorectal carcinogenesis. Allele loss was detected at the DCC (29%), p53 (66%), RB1 (50%) and NF1 (14%) loci and in the APC/MCC region (50%), but not at the WT1 or NM23 loci. These rapid, and mostly gene-specific, fluorescent multiplex PCR assays for allele loss detection could be modified to devise a single molecular diagnostic test for the important lesions in colorectal cancer.

Healthy deliveries from biopsied human embryos

Abstract: Preimplantation genetic diagnosis was performed in 122 embryos obtained by FVF from 11 patients carriers of haemophilia, Duchenne's muscular dystrophy, Barth's syndrome, cystic fibrosis, Pelizaeus-Merzbacher syndrome or Rett's syndrome. After multiplex potymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) analysis with multiple probes, 28 embryos diagnosed as not affected were replaced. Of these, eight implanted (28%) and produced three ongoing pregnancies, three deliveries of four babies and a biochemical pregnancy. However, one case screened for cystic fibrosis was misdiagnosed and the pregnancy was terminated. In order to evaluate the efficiency of multiplex PCR, 55 non-replaced embryos were reassessed by PCR or by FISH. Identical results were obtained in all cases. However, one embryo which had only X-chromosome specific amplification by PCR was found to be XO in all its cells by FISH. Although multiplex PCR is demonstrated to be reliable for sexing of human embryos, FISH has the additional advantages of supplying ploidy assessment while not being affected by contamination .

Detection and Characterization of Atypical Mycobacteria by the Polymerase Chain Reaction

Abstract: The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, and Mycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, including M. kansasii and Mycobacterium leprae, in formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin-embedded tissue sections.

Optimization of Multiplex PCRs

Abstract: The development of the polymerase chain reaction (PCR) has enabled rapid and efficient analysis of specific DNA sequences (Mullis and Faloona, 1987). Most PCR strategies are designed to amplify one or more target sequences with a single set of oligonucleotide primers. However, many experimental approaches require the analysis of a variety of DNA sequences, which necessitates that multiple PCRs be performed on the same or related DNA templates. Considerable savings of time and effort can be achieved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex PCR (Chamberlain et al., 1988, Chamberlain et al., 1989).

Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach.

Abstract: A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.

Thermostable polymerase specific antibody-containing DNA amplification composition and kit

Abstract: Antibodies which are specific to a thermostable DNA polymerase can be used to reduce or eliminate the formation of non-specific products in polymerase chain reaction methods. These antibodies and other temperature sensitive inhibitors are effective to inhibit DNA polymerase enzymatic activity at a certain temperature T 1 which is generally below about 85° C. The inhibitors are irreversibly inactivated at temperature T 2 which is generally above about 40° C. T 2 is also greater than T 1 . Such inhibitors can be supplied individually or in admixture with the DNA polymerase in a diagnostic test kit suitable for PCR.

Amplification of Salmonella Chromosomal DNA Using the Polymerase Chain Reaction

Abstract: A Salmonella-specific polymerase chain reaction (PCR) was developed and standardized. The origin of the primers was a recombinant clone (C7) that contained Salmonella-specific HindIII fragment DNA of 2.1-kilobase pairs. Based on the sequence data of Salmonella enteritidis recombinant clone C7, two primers designated NK1 (21 nucleotides) and NK2 (24 nucleotides) were synthesized for use in the PCR. A Salmonella-specific 2.0-kilobase pair DNA product was amplified by the primers from 23 species of Salmonella, but not from 19 enteric and non-enteric bacteria. As little as 330 fg of Salmonella DNA was detected using either ethidium bromide/ultraviolet exposure of gels or Southern blot hybridization with a C7 clone.

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines.

Abstract: Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2 bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.

Identification of Clavibacter michiganensis subsp. sepedonicus using the polymerase chain reaction

Abstract: From the sequence of a subcloned DNA fragment of a highly conserved plasmid of Clavibacter michiganensis subsp. sepedonicus a pair of oligonucleotides were devised for use as polymerase chain reaction primers. The primer sequences do not show significant homology with any other sequence deposited in public databases. Polymerase chain reactions carried out using this primer pair and untreated cells of all strains of C. michiganesis sepedonicus tested resulted in the amplification of a DNA fragment of about 670 base pairs. No amplification was observed when bacteria belonging to other species were submitted to polymerase chain reaction under the same conditions. The detection limit of the assay was 4 x 10(3) bacteria.

Amplification of strains of bovine herpesvirus 1 by use of polymerase chain reaction with primers in the thymidine kinase region.

Abstract: A primer pair was designed from the published nucleotide sequence of the coding region of the bovine herpesvirus 1 (BHV-1) thymidine kinase (tk) gene for use in detection of the virus by use of polymerase chain reaction (PCR) amplification of 12 BHV-1 strains (3 ATCC and 9 local isolates). A tk deletion mutant BHV-1, and 2 BHV-4 strains from ATCC were used as negative controls. One strain each of feline herpes-virus, equine herpesvirus, and bovine adenovirus, and 2 noninoculated bovine cultured cells--bovine fetal testis and Madin-Darby bovine kidney--also were examined to verify specificity of the primers. A PCR product, 183 bp long, was detected by ethidium bromide staining after agarose gel electrophoresis, when purified DNA from cell cultures infected with BHV-1 strain LA was used as template. Specificity of the PCR product was confirmed by restriction digestion with Sac II enzyme and Southern blot hybridization. Amplification was detected by ethidium bromide staining of agarose gels and/or Southern blot hybridization with the radiolabeled PCR product of the LA strain in similarly prepared DNA templates of 5 other BHV-1 strains, 2 obtained from ATCC and 3 of the 9 local isolates. In a modified PCR protocol, using virus suspensions treated with a nucleic acid-releasing cocktail, substantial amplification was obtained for the 3 BHV-1 strains from ATCC and for all 9 local bovine herpesvirus field isolates.(ABSTRACT TRUNCATED AT 250 WORDS)