Polymerase Chain Reaction Conditions Research Articles

An insertion/deletion polymorphism at the D15S63 locus in the critical Prader-Willi syndrome region in 15q11-13.

Source and description. Prader-Willi syndrome (PWS) is a neurogenetic disorder that is caused by a paternal deletion of 15q 11-13, uniparental maternal disomy of chromosome 15 or imprinting mutations (Nicholls 1993; Reis et al. 1994). At present, the gene for the small nuclear ribonucleoprotein N (SNRPN), which is transcribed from the paternal chromosome only (Glenn at al. 1993; Nakao et al. 1994; Reed and Left 1994), defines the smallest region of deletion overlap (SRO) in PWS (Reis et al. 1994). SNRPN maps 130 kb distal to PW71 (D15S63), which detects methylation specific to the parent-of-origin (Buiting et al. 1993; Dittrich et al. 1993b). Although the methylation test can be used to identify most if not all patients with PWS (Dittrich et al. 1992; Buiting et al. 1994), it gives no information about the nature of the genetic lesion. To distinguish between an imprinting mutation and a deletion, and to determine the extent of a microdeletion, it is necessary to test polymorphic markers around D15S63. Up to now, only two D15S63 po lymorph isms with a low information content have been described (Wagstaff et al. 1993; Dittrich et al. 1993a). Here, we present a common two-allele insertion/deletion polymorphism close to PW71. Yeast artificial chromosome (YAC) B58C7 is a 125-kb YAC containing D15S63 (Kuwano et al. 1992). The right end (YR9C) was isolated by inverse polymerase chain reaction (PCR) (Dittrich et al. 1993b). As YR9C contains part of an Alu sequence, the inner PCR primers yr9a (5"TCGTAGGGAGGTCTAACTTG-3") and yr9b (5"-GAAGAGGCAGTTTGTGATTG-3 ' ) were used to amplify a 495-bp single copy fragment, YR9AB. PCR conditions were as follows: YAC DNA of B58C7 (20 ng) or plasmid DNA of pYR9C (2 pg) was added to a 50-p.1 reaction volume containing 200 gM of each dNTP, 1 gM of each primer, 2.5U AmpliTaq, 1.5mM MgCI2, 50raM KC1, 10 mM TRIS-HC1 pH8.3, 0.1% gelatin. After an initial denaturation for 4min at 94~ the samples underwent 35 cycles of denaturing at 94~ for 30 s, annealing at 58~ for 30 s, extension at 72~ for l min and a final extension at 72~ for 5 min in a Perkin Elmer Thermocycler 9600. The PCR product was gel-purified and used as a probe in Southern blot hybridization.

Rapid diagnosis of cytomegalovirus lung infection by DNA amplification in bronchoalveolar lavages

We used polymerase chain reaction (PCR) to detect cytomegalovirus (CMV) deoxyribonucleic acid (DNA) in the bronchoalveolar lavages (BAL) of 16 CMV-infected patients with active disease. We also tested PCR on a control group of 20 patients including latently infected patients without evidence of active CMV infection. Results were compared with those of CMV culture and of a rapid method of diagnosis which detects CMV in BAL cells by nucleic acid hybridization. PCR allowed the diagnosis in 93% of the actively infected patients compared to 73% for the CMV culture. Among the 20 control patients without evidence of active CMV infection, PCR was negative in all the 24 BAL tested. Hybridization on BAL cells with the CMV probe detected nine out of 10 actively infected patients, but the specificity of the test was only 68·5%. In our experience, PCR appears to be at least as sensitive as CMV culture, it provides results faster and it performs better than the detection of the virus by hybridization on BAL cells. Only active CMV infection was detected with the PCR conditions used in our study. This suggests that the PCR can be applied to bronchoalveolar lavage fluids as a rapid method to detect CMV lung infection.

Analysis of the Vβ specificity of superantigen activation with a rapid and sensitive method using RT PCR and an automatic DNA analyser

Polymerase chain reaction (PCR) by the specific amplification of a DNA target sequence has been shown to permit analysis of T cell receptor usage. The complete repertoire is amplified using oligonucleotide primers specific for each of the known Vα or Vβ regions of the T cell receptor. One of the methods currently used to appreciate the relative quantity of different V chains of the TCR is by coamplifying in the same reaction tube the variable region of one chain together with the constant region of the other chain. We have optimised PCR conditions and analysed PCR products on an automatic DNA analyser facilitating the quantification of the amplified products, avoiding the use of radioisotopes, and allowing the determination of the sizes of CDR3 regions, thus giving new information on the modification of the T cell repertoire. This method was used to analyse the precise Vβ specificity of the T cell activation with the superantigen SEB.

DNA sequence from the SRY gene of the sperm whale (Physeter macrocephalus) for use in molecular sexing

We sequenced a 152 base pair fragment of the sperm whale (Physeter macrocephalus) SRY gene in order to obtain species-specific primers for determination of sex by the polymerase chain reaction (PCR). Sequence identity between the sperm whale SRY fragment and the homologous motif in a variety of other mammals was high, though generally higher with ungulates (~88%) than with the human (85%), rabbit (80%), mouse (75%), or marsupial mouse (66%). When primers based on the sperm whale sequence were employed for PCR, a single product was amplified from male sperm whale DNA, whereas no product was amplified from female DNA. This SRY fragment was also amplified from other cetacean male DNA, but not from human DNA. Thus, under appropriate PCR conditions, the use of the cetacean-specific primers in an assay to determine sex eliminates the possibility of false results owing to contamination by human DNA. To confirm the results of the SRY analysis we sexed the same individual sperm whales by restriction analysis of PCR-amplified fragments from the ZFY and ZFX genes, using universal primers and methods previously reported to work for other cetacean species.

Amplification of Sequences from Affected Individuals

This unit describes methods for obtaining DNA sequences from an individual affected by a genetic disorder. Target sequences that may be present at very low copy number in patient samples are amplified by the polymerase chain reaction (PCR). The first Basic Protocol 1 describes harvesting of mRNA from peripheral lymphocytes, which can even be utilized for genes with tissue-specific patterns of expression. This technique has the advantage of ease of access to appropriate patient samples and also may provide a more efficient means of screening coding sequences than would analysis of individual exons. An alternate protocol describes modifications in PCR conditions that facilitate mutation analysis and sequencing. When the genomic sequence for a given candidate gene is known, the second Basic Protocol 2 may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.

Amplification and Nucleotide Sequences of Ribosomal Protein and 16 S rRNA Genes of Mycoplasma-like Organism Associated with Paulownia Witches' Broom.

To establish a sensitive and reliable detection method of paulownia witches' broom (PWB)-MLO, polymerase chain reactions (PCR) using two primer pairs for ribosomal protein (rp) and 16S rDNA (rD) were conducted. DNA fragments, 1.2kbp for the rp primer pair and 1.3kbp for the rD primer pair, were amplified in DNA samples extracted from infected paulownia leaves but not in healthy leaves, in controlled PCR conditions. Based on the use of both primer pairs, these disease-specific DNAs were detected after amplification of 100pg of total DNA from infected leaves. The nucleotide sequences of amplified ribosomal protein and 16S rRNA genes show that PWB-MLO is closely related to aster yellows type-MLOs.

Selective enrichment of Th1 CD45RBlow CD4+ T cells in autoimmune infiltrates in experimental allergic encephalomyelitis.

The cytokine effector status of CD4+ T cells from lymph nodes (LN) and the central nervous system (CNS) of SJL/J mice immunized with autoantigen in adjuvant for the induction of experimental allergic encephalomyelitis (EAE) was compared. CD4+ T cells were FACS sorted based on the levels of expression of the activation marker CD45RB. Low levels of expression of this surface marker are induced by antigen recognition and are associated with 'effector' T cell function. Reverse transcriptase polymerase chain reaction (PCR) was used to analyze the expression of different T cell cytokine genes in the sorted populations. CD45RBlow cells constituted a minority of CD4+ cells in the LN and expressed elevated levels of IL-2, IFN-gamma, and IL-4 mRNA, whereas the CD45RBlow CD4+ population did not express detectable message for these cytokines under linear PCR conditions. By contrast to the LN, CD4+ cells from the CNS were predominantly CD45RBlow and expressed readily detectable levels of IL-2 and IFN-gamma mRNA, but almost no IL-4 transcription could be detected. IL-4 mRNA levels in CNS were 100- to 250-fold lower than in LN. Also, IL-4 message could not be detected in the CNS 1 week after remission. A cytokine-specific immunocytochemical single cell staining technique was used to enumerate cytokine-producing cells in LN cell populations and in CNS infiltrates. Between 1 and 5% cells in isolated LN cells produced detectable IL-2 and IFN-gamma. By contrast, the frequency of cytokine-producing cells stained in perivascular infiltrates in frozen sections from the brains of animals with active EAE was 10-fold higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Assignment of 30 Microsatellite Loci to the Linkage Map of Arabidopsis

Thirty microsatellite loci were assigned to the Arabidopsis linkage map. Several microsatellite sequences in Arabidopsis DNA were found by searching the EMBL and GenBank databases, and a number of these were subsequently found to detect polymorphisms between different Arabidopsis strains by the polymerase chain reaction (PCR). After the presence of microsatellites in Arabidopsis and their utility for genetic mapping had been demonstrated, systematic screening for (CA)n and (GA)n sequences was carried out on marker-selected plasmid libraries and a small-insert genomic library. Positive clones were sequenced, PCR primers flanking the repeats were synthesized, and PCR was carried out on different strains to look for useful polymorphisms. Surprisingly, of 18 (CA)n repeats (n > 13), only one was polymorphic. In contrast, 25 of 30 (GA)n repeats, 2 of 3 (AT)n repeats, and 2 of 4 (A)n repeats were polymorphic. The majority of the (CA)n repeats were complex, with adjacent short di-, tri-, or tetranucleotide repeats, whereas most of the (GA)n, (TA)n, and (A)n repeats were simple. The (CA)n repeats were also refractory to PCR analysis, requiring extensive optimization of PCR conditions, whereas the other repeat classes were mostly amplified with a single set of standard conditions. When polymorphisms were detected, the microsatellites were mapped using a set of recombinant inbred lines originating from a cross between the strains Columbia and Landsberg erecta.

Improved polymerase fidelity in PCR-SSCPA

Polymerase fidelity is important in any application of the polymerase chain reaction (PCR). In single-strand conformation polymorphism analysis (SSCPA) where one may be looking for a small number of altered DNA strands in the presence of thousands of unchanged strands it is critical. We have examined the effect of PCR conditions, product purification and SSCP analysis on the measured error rates with 4 thermostable polymerases (Taq, Vent, Pyrostase and Pfu). Error rates have been calculated by densitometric scanning of SSCPA gel images. Using PCR conditions which maximize fidelity and eliminating products which include large additions or deletions we have achieved error rates of 10 −5 to 10 −6. Such low rates heighten the probability that relatively infrequent mutations will be identified. Further, the densitometric scanning of gel images provides a useful modification of conventional SSCPA which facilitates such identification.

Production of Reliable Randomly Amplified Polymorphic DNA (RAPD) Markers from DNA of Woody Plants

A procedure for identifying reproducible RAPD markers from woody plant DNA is presented. The procedure relies on using a PCR buffer that contains 1% Triton-X-100 and 0.1 % gelatin [previously described for successful polymerase chain reaction (PCR) amplification of 16S/23S rRNA intergenic spacer regions from eubacteria], and amplification conditions of 50 cycles: 30 sec at 94C, 70 sec at 48C, and 120 sec at 72C. The combination of this buffer and these conditions amplified consistent fragments in higher amounts, as compared to other standard PCR buffers and conditions generally used for RAPD analysis. This procedure resulted in reliable RAPD patterns for all organisms tested. Chemical name used: α-[4-(1,1,3,3,-tetramethylbutyl)phenyl]-cohydroxypoly(oxy-l,2-ethanediyl) (Triton-X-l00).

Regional and Cell-Specific Expression of GDNF in Rat Brain

The survival of ventral mesencephalic substantia nigra (SN) dopamine neurons, which degenerate in Parkinson's disease, is enhanced by glial cells in vitro . The recent isolation of glial cell line-derived growth factor (GDNF), a molecule with apparently selective effects on dopamine (DA) neurons in vitro , raises the question of whether this factor is found in normal brain cells. In this study, the polymerase chain reaction (PCR) was employed to determine the regional distribution and cellular localization of GDNF in the rat central nervous system. GDNF was expressed by SN and basal forebrain Type 1 (T1) astrocytes, with trace transcript levels present in cortical T1 astrocytes. Neuronal cultures of embryonic SN also expressed GDNF. Regionally, postnatal striatum contained the highest GDNF mRNA levels in vivo under the PCR conditions employed. Our data suggest a role for GDNF in both local and target-derived support of DA neurons, as well as potential involvement in the support of other neuronal populations in vivo.

Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.

Sex Determination on Samples of Bovine Meat by Polymerase Chain Reaction

ABSTRACTA simple method for determining sex on samples of bovine meat is based on the polymerase chain reaction (PCR) amplification of bovine Y chromosome‐specific sequences using total DNA from meat as template. Under optimized PCR conditions, an amplified DNA fragment, with size between 298 bp and 344 bp markers, was detected only in the presence of male meat DNA as template. The procedure, including DNA extraction, PCR amplification and electrophoretic analysis, required about 5 hr and could be carried out starting from fresh or frozen beef. Unambiguous results were obtained in the presence of amounts of template DNA ranging from 0.01 to 100 ng. This procedure has potential application in regulatory analysis to specifically differentiate sex origination of meat samples.

Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA

A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.

PARVOVIRUS B19 INFECTION CAUSING PURE RED CELL APLASIA IN A RECIPIENT OF PEDIATRIC DONOR KIDNEYS1

PARVOVIRUS B19 INFECTION CAUSING PURE RED CELL APLASIA IN A RECIPIENT OF PEDIATRIC DONOR KIDNEYS1

DNA hybridization and polymerase chain reaction (PCR) tests for identification of Gaeumannomyces, Phialophora and Magnaporthe isolates

DNA hybridization and PCR tests for Gaeumannomyces graminis and related fungi were developed to aid in their identification and classification. Hybridizations with a Gaeumannomyces graminis var. tritici mitochondrial DNA probe to restriction fragments from other Gaeumannomyces, Phialophora or Magnaporthe isolates demonstrated that almost all of the tested Gaeumannomyces and Phialophora isolates, including Gaeumannomyces cylindrosporus and Gaeumannomyces incrustans , had sequences homologous to the probe. In addition, M. poae had strong homology with the probe. Most isolates of Gaeumannomyces graminis var. tritici , the causative agent of take-all disease of cereals, had identical Eco R I and Msp I hybridization patterns, whereas sizes of hybridizing restriction fragments of Phialophora and Magnaporthe isolates showed more variation. Polymerase chain reaction (PCR) tests with primers developed for G. graminis var. tritici were positive for all G. graminis isolates. PCR tests for G. incrustans, G. cylindrosporus, Phialophora and Magnaporthe were positive, but test conditions in which positive results were obtained were more limited than PCR conditions which amplified G. graminis DNA.

A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus

We have developed a polymerase chain reaction (PCR)-directed sequencing strategy for rapid sequencing of DNA from crude viral or cell preparations. The basic strategy consists of two phases. In the first phase, the target DNA is amplified by symmetric PCR with low concentrations of deoxyribonucleotide triphosphate (dNTP) and oligodeoxyribonucleotide primers. This results is exponential amplification of DNA in the initial cycles, reaching a plateau by 25 cycles due to limiting concentrations of dNTP and primers. In the second phase, a small aliquot of the PCR mixture is amplified without any purification, by asymmetric PCR in the presence of a 5′-labeled primer and one of the four dideoxyribonucleotide triphosphates. This results in the accumulation of single-stranded DNA products that are terminated at specific points by incorporation of the appropriate dideoxyribonucleotide monophosphate. The products are then analyzed by electrophoresis on a sequencing gel followed by autoradiography. The PCR conditions are optimized to generate sequence ladders of several hundred nucleotides starting from as low as 100 copies of bacteriophage or bacterial genome in one to two days.

Polymorphisms revealed by PCR with single, short-sized, arbitrary primers are reliable markers for mouse and rat gene mapping.

Ten single, arbitrarily designed oligodeoxynucleotide primers, with 50-70% (G+C) content, were used to amplify by polymerase chain reaction (PCR) sequences with DNA templates from several mouse species (Mus spretus, Mus musculus musculus, and Mus musculus domesticus), as well as DNA from the laboratory rat (Rattus norvegicus). Eight of these ten primers, used either individually or associated in pairs, generated a total of 13 polymorphic products which were used as genetic markers. All of these polymorphic sequences but one were mapped to a particular mouse chromosome, by use of DNA panels prepared either from interspecific backcross progeny of the type (C57BL/6 x Mus spretus)F1 x C57BL/6 or DNA samples prepared from two sets of recombinant inbred (RI) strains (AKXL and BXD). Six rat-specific DNA segments were also assigned to a particular chromosome with DNA panels prepared from 18 rat/mouse somatic cell hybrids segregating rat chromosomes. From these experiments we conclude that, under precisely standardized PCR conditions, the DNA molecules amplified with these arbitrarily designed primers are useful and reliable markers for genetic mapping in both mouse and rat.

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

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.

A Polymerase Chain Reaction (PCR) Method for Sex and Species Determination with Novel Controls for Deoxyribonucleic Acid (DNA) Template Length

Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.