Showing papers on "Multiplex polymerase chain reaction published in 1989"

Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction

Abstract: Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.

Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

Abstract: A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

Improved double-stranded DNA sequencing using the linear polymerase chain reaction.

Abstract: One of the problems associated with double-3tranded DNA sequencing is that most of the [5'-3P]-labelied oligonuoleotide primer i3 not extended by the DNA polymerase. Hence, most of the labelled primer is wasted and gels have to be autoradiographed for several days. In the method described in this paper, almost all of the labelled primer is extended by the DNA polymerase and thus a shorter autoradiograph exposure is needed. This method Is a modification of the linear polymerase chain reaction (LPCR) where only one o ligonucleotide 13 present (1). It U363 Taq DNA polymerase and, since polymerisation is at 72°C, results in a low level of sequencing artifacts. Multiple LPCR cycle3 also removes sequencing artifacts. The use of lower than normal dNTP concentrations i3 necessary to achieve

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

Abstract: The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Detection of single DNA base differences by competitive oligonucleotide priming

Abstract: Synthetic DNA oligonucleotides can serve as efficient primers for DNA synthesis even when there is a single base mismatch between the primers and the corresponding DNA template. However, when the primer-template annealing is carried out with a mixture of primers and at low stringency the binding of a perfectly matched primer is strongly favored relative to a primer differing by a single base. This primer competition is observed over a range of oligonucleotide sizes from twelve to sixteen bases and with a variety of base mismatches. When coupled with the polymerase chain reaction, for the amplification of specific DNA sequences, competitive oligonucleotide priming provides a simple general strategy for the detection of single DNA base differences.

Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction.

Abstract: By using a single synthetic oligonucleotide primer pair in the polymerase chain reaction, we amplified specific Shiga-like-toxin (SLT) gene segments from DNAs of 20 clinical Escherichia coli isolates, irrespective of whether they produce SLT-I, SLT-II, or heretofore uncategorized SLTs. These segments were not detectable in any of 20 nontoxigenic E. coli strains. The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; i.e., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes. In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II DNA sequences. Amplified sequences of target DNA obtained by polymerase chain reaction were visualized after gel electrophoresis by ethidium bromide staining, and definitive identification of the amplification product as an SLT gene segment was achieved by hybridization to SLT-I- and SLT-II-specific 20-base oligonucleotide probes complementary to a portion of the amplified sequences but not to the primers. The detecting oligonucleotide probes shared only 30% base homology and were shown to recognize specifically SLT-I or SLT-II sequences within genomic DNA. Moreover, they were used to distinguish whether the amplified sequence originated from SLT-I or SLT-II genes. The PCR system with the primers described here is a powerful technique to amplify SLT sequences in E. coli strains that produce serologically distinct SLTs and will facilitate identification of these pathogens, particularly among a multitude of nonpathogenic E. coli strains. Images

Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.

Abstract: We have developed a rapid procedure for the detection of serum hepatitis B virus (HBV) DNA using the polymerase chain reaction (PCR) technique. HBV DNA is released from virions by incubating serum with 0.1 M NaOH for 60 min at 37 degrees C. The mixture is brought to neutral pH with HCl, and the HBV DNA sequences are detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with two successive sets of primer pairs. The detection limit of this method (i.e., 10(-5) pg of HBV DNA) is equivalent to that previously determined by one round of PCR amplification and Southern blot hybridization analysis. The advantages are that the assay can be completed in 1 day, is very sensitive, and does not require the use of radiolabeled reagents. Images

Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny

Abstract: We describe the simultaneous amplification of different segments of foreign DNA in transgenic plants using the polymerase chain reaction (PCR). We used PCR to simultaneously amplify different regions of transformed T-DNA in order to assay the integrity of transformed constructions in primary tomato transformants. We also used simultaneous PCR amplification to examine the segregation of transformed sequences in progeny of primary transformants. A tomato transformant containing the maize transposable elementAc was crossed to transformants containing the non-autonomousDs1 element flanked by maizeAdh1 sequences. We then ran PCR reactions on DNA from F1 progeny using two sets of primers, one set homologous toAc and one set homologous toAdh1 sequences on either side ofDs1. Because theAc andAdh1 primers resulted in amplification of fragments of different sizes, it was possible to monitor the inheritance ofAc and theDs1 containingAdh1 genein a single reaction. Additionally, it was possible to identify F1 plants in whichDs1 had excised by the amplification of a fragment the size predicted for an empty donor site. In order to run these reactions, we have constructed a simple and inexpensive thermal cycler which, when used in conjunction with the rapid miniscreen plant DNA isolation procedure described, allows the processing of a large number of samples in a single day. Therefore, we have shown that PCR can be a useful tool to monitor the integrity of foreign genes in transgenic plants, to follow the segregation of foreign DNA in progeny, and to assay for the excision of transposable elements.

Detection of amplified oncogenes by differential polymerase chain reaction.

Abstract: Oncogene amplification has been found in a variety of human cancers and may have prognostic importance. Therefore, techniques which facilitate detection of gene amplification could have wide applicability. We have devised a sensitive, rapid, and non-radioactive procedure for detecting alterations in gene copy number based on the polymerase chain reaction (PCR). In this technique, called differential PCR, a target gene and a single-copy reference gene are co-amplified by PCR in the same reaction vessel. The level of target gene amplification is reflected in the ratio between the two resulting PCR-product bands. We show that this method can detect as low as two-fold amplification of specific target genes. Furthermore, amplification of neu and the epidermal growth factor receptor gene could be detected in as few as 100 breast carcinoma cells or in single sections of formalin-fixed, embedded material.

Methods for generating single stranded dna by the polymerase chain reaction

Abstract: Single stranded DNA can be generated by the polymerase chain reaction using two oligonucleotide primers, one persent in a limiting concentration. The single stranded DNA is useful in procedures involving utilizing nucleic acid probes and for purposes of nucleic acid sequencing.

Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species

Abstract: The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.

A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction.

Abstract: The alphoid satellite family is the only repetitive DNA family showing chromosome specificity. We have developed a simple, rapid, and reliable test for sex diagnosis based on detection of these sequences in undigested genomic DNA using the polymerase chain reaction. In our test, dried blood specimens were the source of DNA. When female DNA was used as a template for the reaction, only the expected 130-bp X-chromosome-specific fragment was detected, while with male DNA both the expected 170-bp Y-chromosome-specific and X-chromosome-specific fragments were detected. The Y-chromosome-specific fragment was further characterized by restriction enzyme analysis. The Y fragment was detectable when DNA obtained from an equivalent of 10 μl of spotted blood was used in the reaction, whereas detection of the X fragment was possible with DNA from an equivalent of 5 μl of blood. Our test may find various applications in newborn screening and in forensic science.

Compositions for and detection of human papillomavirus by specific oligonucleotide polymerase primers using the polymerase chain reaction

Abstract: The presence of human papillomavirus (HPV) in a sample can be detected and the HPV typed by a method that involves the amplification of HPV DNA sequences by the polymerase chain reaction (PCR). The primers used in the method are consensus primers that can be used to amplify a particular region of the genome of any HPV. The presence of HPV in a sample is indicated by the formation of amplified DNA. The HPV is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

Sequencing dna; a modification of the polymerase chain reaction

Abstract: A new process has been developed which facilitates separation of DNA strands following the polymerase chain reaction. The process comprises 1) reacting a double stranded DNA with a biotinylated primer and a non-biotinylated primer to provide a double-stranded DNA wherein one strand has biotin bound to the extension primer; 2) reacting the product of step 1 with a support that will bind the biotin moiety; 3) denaturing the DNA; and 4) separating the amplified single-stranded DNA lacking the biotin moiety from the mixture containing the amplified DNA having biotin bound thereto.