Showing papers on "Multiplex polymerase chain reaction published in 1991"

Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction

Abstract: The polymerase chain reaction (PCR) can be used to detect polymorphisms in the length of amplified sequences between the annealing sites of two synthetic DNA primers. When the distance varies between two individuals then the banding pattern generated by the PCR reaction is essentially a genetic polymorphism and can be mapped in the same way as other genetic markers. This procedure has been used in a number of eukaryotes. Here we report the use of PCR to detect genetic polymorphisms in cereals. Known gene sequences can be used to design primers and detect polymorphic PCR products. This is demonstrated with primers to the α-amylase gene family. A second approach is to use semi-random primers to target diverse regions of the genome. For this purpose the consensus sequences at the intron-exon splice junctions were used. The targeting of the intronexon splice junctions in conjunction with primers of random and defined sequences, such as α-amylase, provides a source of extensive variation in PCR products. These polymorphisms can be mapped as standard genetic markers.

Isolation of simple‐sequence loci for use in polymerase chain reaction‐based DNA fingerprinting

Abstract: Simple sequences are short regions of tandem repetitions of mono-, di-, tri-, or tetranucleotide motifs and occur as repetitive elements in all eukaryotic genomes. These regions tend to be hypervariable in length and can therefore be exploited for DNA fingerprinting purposes, using the polymerase chain reaction with primers flanking such regions. We describe how suitable simple-sequence loci can be isolated from any given eukaryotic DNA. We show the DNA sequences for a number of variants of such loci and discuss the current results on their usefulness for DNA fingerprinting.

Sex identification by polymerase chain reaction using X‐Y homologous primer

Abstract: A method of sex identification using the polymerase chain reaction technique is described. Using a pair of nucleotide primers from an X-Y homologous region, both the X and the Y sequences can be amplified simultaneously, and more importantly, they result in fragments of different lengths. The success of the procedure is therefore monitored by the presence of a X-specific band while sex is identified by the presence or absence of a Y-specific band.

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.

Abstract: Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli.

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

Abstract: In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.

A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods.

Abstract: Existing reactions for the multiplex PCR amplification of exons in the dystrophin gene have been modified to produce two multiplex reactions which separately cover the 5' and 3' major deletion 'hotspots' in the gene, and together detect approximately 98% of all deletions detectable by Southern cDNA hybridisation. A comparative study of 148 patients showed mistypings in both the cDNA hybridisation data (4%) and the PCR analysis (1.2%). We suggest means of circumventing the underlying problems in order to avoid mistyping and subsequent misdiagnosis, and conclude that, with appropriate precautions, multiplex PCR amplification can be the method of choice for detecting deletions in the dystrophin gene.

A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction.

Abstract: We have developed a novel polymerase chain reaction (PCR) method that permits the rapid generation of site-specific mutants and recombinant DNA constructs with a minimum number of steps and primers. DNA segments are modified by using amplifying primers that add homologous ends to the polymerase chain reaction product(s). These homologous ends undergo recombination in vivo following transformation of recA-E. coli strains used routinely in cloning. In vivo circularization of PCR products containing plasmid sequences with a selective marker permits the rapid cloning of the desired mutant or recombinant. In the mutagenesis protocol, 7 of the 12 clones contained the product of interest, and 6 of these clones had no detected error (50% of the clones without detected errors). In each of several recombination protocols, at least 50% of the clones tested contained the insert of interest without detected errors.

Validation Studies on the Analysis of the HLA DQα Locus Using the Polymerase Chain Reaction

Abstract: A series of experiments has been performed to evaluate typing of the HLA DQ alpha gene by polymerase chain reaction (PCR) amplification of the gene and subsequent hybridization with sequence-specific oligonucleotide probes. These experiments were designed to evaluate DQ alpha typing for analysis of evidentiary specimens. Bloodstains were exposed to a variety of conditions and environmental insults. These conditions included exposure to many different types of substrates, various microorganisms that could be encountered in evidentiary stains, sunlight, and a variety of chemical contaminants. Varying amounts of genomic deoxyribonucleic acid (DNA) were amplified to test the sensitivity of DQ alpha typing. The sensitivity of the PCR technique raises the concern that DNA from sources other than the evidentiary material could be detected. A series of experiments was done to evaluate the question of DNA contamination. Purified DNA samples with different DQ alpha types were mixed in different ratios to determine the ratio at which it could not be determined whether an allele was from the sample or the contaminant. Samples were exposed to a variety of situations that could lead to contamination, such as extensive handling and exposure to coughing or sweaty clothing, to other wet bloodstains, and to saliva. The DQ alpha types were determined from 469 individuals from three sample populations (Caucasian, black, and Hispanic), and the genotype frequencies were compared with frequencies previously reported by others. DNA samples from old cases [which had previously been analyzed by restriction fragment length polymorphism (RFLP) typing of variable number of tandem repeat sequences] were typed. All samples that were excluded by DQ alpha typing were also excluded by RFLP analysis, and all samples that were included by RFLP analysis were included by DQ alpha typing. Finally, the problem of allele dropout, or the failure to detect particular alleles, was noted and alleviated by performing the typing under appropriate conditions. The results of these validation experiments indicate that typing of the DQ alpha gene by PCR and detection of specific alleles can be accomplished, when the typing is done using proper protocols, without producing false positive or false negative results.

Polymerase chain reaction for detection of Mycoplasma gallisepticum.

Abstract: A species-specific 760-base pair (bp) BamHI to EcoRI DNA fragment (fMG-2) was isolated from a Mycoplasma gallisepticum (MG) genomic library constructed in plasmid pUC8. Based on the DNA sequence data of fMG-2, a pair of 25 base primers, designated amplification (Amp) left (L) and right (R) primers, was synthesized. When used in the polymerase chain reaction (PCR), the Amp L and R primers directed amplification of DNA of 16 MG strains yielding an expected 732-bp product, but did not amplify DNA of Escherichia coli, calf thymus, lambda phage, pUC8 plasmid, or 16 other species of avian mycoplasmas. As low as 10(-6) picogram of MG DNA, a fraction of the total chromosomal content of one cell, was detected following amplification by PCR. PCR amplification products were visualized by either ethidium bromide/ultraviolet exposure or hybridization with a 481-bp probe (fMG-3) prepared from the central region of fMG-2.

K-tuple frequency in the human genome and polymerase chain reaction.

Abstract: The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

Abstract: The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.

Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction.

Abstract: A polymerase chain reaction system for the detection of varicella-zoster virus was established. Of 25 nucleotides, 4 oligonucleotide pairs (regions of thymidine kinase, thymidylate synthetase, glycoprotein I, and immediate early gene) were synthesized. The first three oligonucleotide pairs could be used as primers on the basis of specific DNA amplification. Varicella-zoster virus DNA was amplified by this polymerase chain reaction system in 20 of 20 vesicle samples, 5 of 6 crusts, and 12 of 13 throat swabs collected from patients with clinical varicella.

A polymerase chain reaction mediated by a single primer : cloning of genomic sequences adjacent to a serotonin receptor protein coding region

Abstract: Under appropriate conditions, specific double-stranded DNA product was generated after amplification of genomic DNA sequences in a polymerase chain-like reaction that contained only a single primer. This type of amplification reaction was performed with a variety of primers and substrate DNAs. In addition to nonspecific heterogeneous products, 5 of 11 primers reproducibly directed synthesis of double-stranded DNA that corresponded to the region of the template that contained the authentic primer annealing site. Three of these amplified products were cloned and their ends were sequenced. All three contained a copy of the primer at both 5' ends, and the position of one of the primers represented the authentic primer binding site. In each case, the location of the second copy of the primer indicated that it had initially hybridized to a partially homologous sequence in the template DNA. This single primer reaction makes it possible to amplify and clone a DNA region of unknown sequence that is adjacent to a known DNA sequence. One of the single primer reaction products described here included sequence to the 5' side of the coding region of a serotonin receptor gene that contained a functional promoter.

Automated amplification and sequencing of human mitochondrial DNA.

Abstract: Part of the human mitochondrial D-loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M13-21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single-stranded DNA was generated. This was then sequenced directly by the diodeoxy chain termination method using dye-labelled universal sequencing primers in conjunction with a fluorescence-based DNA sequencer. This enabled a 403-base-pair hypervariable segment of the D-loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.

The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction.

Abstract: A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.

Multiple sclerosis, retroviruses, and PCR

Abstract: Previously reported serologic and polymerase chain reaction (PCR)-based findings have suggested an association between the human retrovirus, HTLV-I, and multiple sclerosis (MS). Due to the inherent ability of PCR to produce false-positive results, we developed a set of physical and procedural safeguards to minimize the possibility of molecular carryover. These were applied as part of a blinded, large-scale, multipopulation, multiplex PCR-based study designed to examine this issue of association. Our results do not support the hypothesis that HTLV-I, which plays a role in the pathogenesis of an encephalomyeloneuropathy, HTLV-II, or closely related agents are associated with MS. A concomitant review of the current literature supports this view.

Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.

Abstract: We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.

Decontaminating the polymerase chain reaction.

Abstract: Template DNA, which was added to a modified polymerase chain reaction mixture to simulate contamination, was inactivated by restriction enzyme digestion. After inactivation of the restriction enzymes, additional template, buffer and Taq polymerase were added to the reaction and the polymerase chain reaction proceeded. Results are presented using several different restriction enzymes with four base recognition sequences. The limitations of the method are discussed to indicate the tactics to be used for other DNA sequences.

Detection of bovine viral diarrhea virus, using degenerate oligonucleotide primers and the polymerase chain reaction.

Abstract: A technique for detection of bovine viral diarrhea virus (BVDV) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of BVDV and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral RNA in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of BVDV by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.