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Touchdown PCR - Wikipedia, the free encyclopedia Touchdown PCR or Touchdown style PCR is a method of PCR (polymerase chain reaction) by which degenerate primers will avoid amplifying nonspecific sequence. Touchdown PCR was first reported in Nucleic Acids Research. "Touchdown' PCR to circumvent spurious priming during gene amplification." Nucleic Acids Res. en.wikipedia.org /wiki/Touchdown_PCR   (308 words)

Polymerase chain reaction - Wikipedia, the free encyclopedia PCR is often used to amplify the gene, which can then be inserted into a vector (a vector is a piece of DNA which 'carries' the gene into the GEO) such as a plasmid (a circular DNA molecule) (Fig. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar. Later, this original PCR process was greatly improved by the use of DNA polymerase taken from thermophilic bacteria grown in geysers at a temperature of over 110°C (230°F). www.wikipedia.org /wiki/Polymerase_chain_reaction   (3029 words)

BioMed Central | Full text | A touchdown nucleic acid amplification protocol as an alternative to culture backup for ... Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming [14]. PCR identified one or more viruses in specimens from 84 of the 183 patients and the 4 NEQAS positive specimens, detecting a total of 99 viruses as shown in Table 1. Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets [19-22], its use in this study has extended its role further and in so doing brought closer the goal of undertaking molecular diagnostics in a routine setting. www.biomedcentral.com /1471-2180/4/41   (5012 words)

A Sensitive, Non-nested Touchdown PCR for Same Day Detection of Chlamydia pneumoniae in Clinical Specimens.   (Site not responding. Last check: 2007-10-14) Methods: Using the nested Tong and Sillis PCR as a reference standard, we determined the sensitivity and specificity of a new touchdown PCR that uses a single round of amplification with Tong & Sillis’ primers targeting the ompA gene and a microtitre plate bioluminescence hybridization assay (ChemFLASH, Chemicon) for detection. Initially, the 2 PCRs were compared using 10 replicates of 10 serial dilutions of a stock of C. Results: The non-nested PCR with bioluminescence detection was equally sensitive to the nested PCR. www.cacmid.ca /abstracts/a56.html   (284 words)

Polymerase chain reaction - free-definition   (Site not responding. Last check: 2007-10-14) Polymerase Chain Reaction (PCR) is a molecular biological method for amplifying (creating multiple copies of) DNA without using a living organism, such as E. The basic method for performing PCR was invented by Kary Mullis, who was awarded the Nobel Prize in Chemistry in October 1993 for this achievement, only seven years after he first published his ideas. PCR is often used to amplify the gene, which can then be inserted into a vector (a vector is a means of inserting a gene into an organism) such as a plasmid (a circular DNA molecule) (Fig. www.free-definition.com /Polymerase-chain-reaction.html   (2583 words)

Methods - Quantitative pcr for Chlamydia Quantitative PCR offers significant advantages over conventional pcr in its rapidity, the ease with which it can be automated, the potential decreased risk of carry-over contamination and the provision of a quantitative result. Of 19 serology-positive patients, 14 were confirmed by the multiplex PCR to be infected by either one of the three pathogens. Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry. www.chlamydiae.com /restricted/docs/Methods/tech_quantitative_pcr.asp   (1597 words)

The NFL Directory: touchdown pcr   (Site not responding. Last check: 2007-10-14) Touchdown PCR is an easy way to cut down on stuttering and non... Touchdown PCR involves decreasing the annealling temperature by 1 degree C every second... 'Touchdown' PCR to circumvent spurious priming during gene amplification. www.thenfldirectory.com /25/touchdown-pcr-.html   (671 words)

Biocompare - Product Literature Center RNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer. The second-round PCR is performed with two new primers that hybridize to sequences internal to the first-round primer—target sequences. The kit is highly suited for multiplex PCR applications such as typing of genetically modified animals and plants, microsatellite analysis, determination of bacteria and viruses, or amplification of regions carrying SNPs. www.biocompare.com /techart.asp?id=680   (5671 words)

Brinkmann Instruments Canada ::: Using the TripleMaster PCR System for Robust Amplification of GC-Rich DNA Templates   (Site not responding. Last check: 2007-10-14) Touchdown PCR was then employed to prove this method is capable of achieving optimal amplification and specificity without the need for thermal cycling optimization experiments. During touchdown PCR, the annealing temperature of the first cycle is set at or a few degrees above the melting temperatures of the primers. Touchdown PCR is a valuable method for generating robust PCR reactions without time-consuming optimization experiments. www.brinkmanncan.com /applications/PCR_appl_3xmaster-gc-rich.asp   (1721 words)

BioMed Central | Full text | The human homologue of unc-93 maps to chromosome 6q27 – characterisation and ... In total, exons 1, 2, 5, 8 from the 36 tumour samples were amplified by primers flanking the exons using the "touchdown" programme [29] and subjected to the analysis on the WAVE machine. Individual exons were amplified by PCR from genomic DNA from a panel of 36 malignant tumours using primers labelled with HEX (green, forward primers), and FAM (blue, reverse primers) and applied to MDE gel on a ABI 377 system (PE Applied Biosystems). 'Touchdown' PCR [29] was performed to amplify individual exons. www.biomedcentral.com /1471-2156/3/20   (6697 words)

Haematologica - e-cases PCR monitoring is of prognostic value when used for minimal residual disease (MRD) evaluation during the follow-up. It is obvious that a PCR technique should be attempted in order to establish a precise diagnosis especially in cases when the histology or other laboratory tests are not conclusive. We recently presented that rituximab alone or in a combination with chemotherapy may induce PCR negativity in the BM of high percentage of NHL patients and that is connected with a better prognosis. www.haematologica.org /e-cases/2003_03/06.htm   (1459 words)

Touchdown PCR | TutorGig.co.uk Encyclopedia   (Site not responding. Last check: 2007-10-14) Its original description can be found in the following article: :RH Don, PT Cox, BJ Wainwright, K Baker, and JS Mattick. Search for Touchdown PCR in Usenet Discussion Groups A Low-cost Approach to PCR: Appropriate Transfer of Biomolecular Techn.. www.tutorgig.co.uk /encyclopedia/getdefn.jsp?keywords=Touchdown_PCR   (359 words)

Wheat SNP Development We are not aware of any IP on this method (beyond PCR) and it is the simplest method we could come up with. PCR with the locus primers and SNP primers will generate bands that have one of two sizes, according to which SNP primer was incorporated, and hence indicating the base in the original templates. In our experience a touchdown PCR of 3 cycles per degree, from 60-55 anneal, then cycle at 55C anneal, tends to generate OK results, but we may revise our opinion after more experience. wheat.pw.usda.gov /ITMI/WheatSNP/methods.html   (275 words)

Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples. Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples. In routine clinical use, easy sample processing and single-round PCR are needed to ensure rapid analysis and to reduce the risk of contamination. We developed a single-round Touchdown PCR (TD-PCR) protocol with the ability to detect PCR inhibition in the specimen. www.aegis.com /aidsline/1998/dec/M98C7297.html   (353 words)

Bringing Genomics to the Wheat Fields - model Resistance   (Site not responding. Last check: 2007-10-14) "Touchdown" PCR, was used to amplify genomic DNA containing both Lr29 and Lr25 resistance genes. The PCR conditions were as described except that the annealing temperature was 65°C for the first 15 cycles and 58°C for the next 25 cycles. Conditions presented here should be consider only as a starting point of the PCR optimization for individual laboratories. maswheat.ucdavis.edu /protocols/Lr29/Lr29_methods.htm   (199 words)

Kennedy, Mol Vis 1998; 4:14. The genomic organization of the mouse CRALBP gene is shown in Figure 2 and the DNA sequence of the gene in Figure 3. The lengths of mouse CRALBP intron 3 (1.8 kb), intron 4 (1.5 kb), intron 5 (2.6 kb) and intron 6 (1.2 kb) were determined by PCR amplification using as a template either the 13 kb [lambda]-phage clone or mouse 129/SvJ genomic DNA (Figure 4). The structure of the mouse CRALBP gene was determined from a clone isolated from a 129/Sv mouse genomic library and from PCR amplification of mouse eye cDNA and mouse 129/SvJ genomic DNA. www.molvis.org /molvis/v4/a14   (3856 words)

Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. ... PCR protocol (14) which did not use a TETR protocol. PCR was 90% (18 of 20) sensitive and 100% (30 of 30) specific. Yamamoto, Y. PCR in Diagnosis of Infection: Detection of Bacteria in Cerebrospinal Fluids. jcm.asm.org /cgi/content/full/38/3/1085   (5612 words)

Utility of Random Amplified Polymorphic DNA PCR and TaqMan Automated Detection in Molecular Identification of ... fumigatus or Neosartorya spp.) amplified by RAPD-PCR with the OPZ19 primer (A), by conventional PCR with primers Z19-276 and Z19-660 (B), or by touchdown PCR with primers Z19-276 and Z19-660 (C). RAPD-PCR and the TaqMan PCR assay appear to be useful strategies for identifying and detecting diagnostic DNA targets. Killgore, G. E., Holloway, B., Tenover, F. A 5' Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the mecA Gene in Staphylococci. jcm.asm.org /cgi/content/full/36/7/2057   (4035 words)

Sequence tagged microsatellite profiling (STMP): improved isolation of DNA sequence flanking target SSRs -- Hayden et ... PCR amplification was performed for 25 cycles (60 s at 92°C, 60 s at 56°C and 60 s at 72°C). A touchdown PCR profile of 32 cycles was used for amplification: Each PCR was performed in replicate using six independently prepared unenriched (UE) and SSR-enriched (E) amplicon pools as template DNA. nar.oupjournals.org /cgi/content/full/30/23/e129   (2461 words)

Ac Protocols If the desired amplified sequence is smaller than 5 kb, a touchdown inverse PCR reaction with a regular Taq polymerase can be used to amplify the fragment in a first round of PCR, followed by a second, nested PCR reaction. The long-distance PCR can be used for both smaller fragments (~ 2 kb), and fragments up to at least ~8 kb in size. A second round of nested PCR is not necessary. bti.cornell.edu /Brutnell_lab2/Projects/Tagging/BMGG_pro_iPCR.html   (1101 words)

PG-IV: P50 - Homologous SSR Markers and SSR-PCR Fingerprinting in Two Pine Genomes   (Site not responding. Last check: 2007-10-14) Of the 22 markers tested, 14 amplified at least 1 locus in loblolly pine under non optimized touchdown PCR conditions. Optimized PCR conditions resulted in stronger and simpler banding patterns, making many of the loci interpretable as genetic markers in loblolly pine. Banding patterns varied widely depending on the repeat motif and anchor, but, in general, the numbers of amplified bands were correlated with SSR frequencies, as determined by plaque hybridization. www.intl-pag.org /pag/4/abstracts/p50.html   (281 words)

[No title] For G. etunicatum we used Advantage(-HF 2 PCR Kit (Clontech, Palo Alto, CA), (touchdown 15 cycles: 94(C for 15 sec, 60(C (-0.5C per cycle) for 15 sec, 68C for 1 min, 10 cycles: 94(C for 15 sec, 53(C for 15 sec, 68(C for 1 min). For G. intraradices (3 randomly selected spores from a clonal culture to assess the intrasporal variation) we used PfuTurbo(DNA polymerase (touchdown 15 cycles: 94(C for 15 sec, 60(C (-0.5(C per cycle) for 15 sec, 75(C for 1 min, 20 cycles: 94(C for 15 sec, 53(C for 15 sec, 75(C for 1 min). PCR amplicons were cloned with TOPO TA Cloning(Kit for Sequencing (Invitrogen, Carlsbad, CA) after QIAquick® PCR purification (Qiagen, Valencia, CA) for amplifications from individual nuclei, and addition of 3’ A-overhangs in all reactions. www.nature.com /nature/journal/v427/n6976/extref/nature02290-s1.doc   (1195 words)

The Nucleotide Sequence of Koala (Phascolarctos cinereus) Retrovirus: a Novel Type C Endogenous Virus Related to Gibbon ... Initial PCR amplification of a 3-kb koala retrovirus (KoRV) sequence from genomic DNA was accomplished with an upstream primer, Touchdown PCR (46) was performed in 50-µl reaction mixtures using the Expand Long Template PCR system (Boehringer, Mannheim) PCR bands were resolved on a 0.9% agarose gel and then excised and extracted with the Qiaquick Gel Extraction kit (Qiagen). jvi.asm.org /cgi/content/full/74/9/4264   (6064 words)

Comparative Study of the Presence of Chlamydia pneumoniae in Cerebrospinal Fluid of Patients with Clinically Definite ... PCR amplification of the major outer membrane protein (MOMP) gene of C. PCR amplification of the MOMP gene of C. a nested-PCR or touchdown PCR assay for the detection of C. cdli.asm.org /cgi/content/full/9/6/1332   (3506 words)

Improved Repetitive-Element PCR Fingerprinting of Salmonella enterica with the Use of Extremely Elevated Annealing ... Improved Repetitive-Element PCR Fingerprinting of Salmonella enterica with the Use of Extremely Elevated Annealing Temperatures -- Johnson and Clabots 7 (2): 258 -- Clinical and Diagnostic Laboratory Immunology PCR products were electrophoresed in 1.0% agarose gels, stained with ethidium bromide, and visualized using a UV transilluminator Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak. cdli.asm.org /cgi/content/full/7/2/258   (3764 words)

The World Famous PCR Jump Station: Polymerase Chain Reaction Your work will be published in The PCR Encyclopedia and will be available, free, to scientists worldwide. Real Time PCR Group New discussion group for PCR using fluorescence based detection, but general PCR questions can be also addressed. Genamics SoftwareSeek is a respository and database of freely- distributable and commercial tools for use in molecular biology and biochemistry. www.highveld.com /pages/pcr.html   (1203 words)

Molecular Biology - How to remove PCR inhibitors from DNA Hi, So many things influence PCR reaction so you can not say there are PCR inhibitors in your DNA samples. If you have suitable DNA control from any kit, you may try it first to explore the PCR condition for this pair of primers. What you describe has happened to me simply because I was solubilizing my maxipreps in TE buffer and was presumably chelating out the magnesium needed for the PCR. www.protocol-online.org /archive/posts/4105.html   (290 words)

PCR SMEARING We have been getting a lot of smearing in our PCR reactions. Smear problems in PCR is most usually a bad ratio of Mg concentration vs. DNA (template + oligos), because Mg ions are also bound to DNA. Although PCR optimizing has been done, the problem still there.So, is the culture cell storage period affect the PCR afficacy,or some compounds produced from the cells prohibit from the targets amplified?i will appreciate you if you can give me any helpful suggestion. www.biowww.net /detail-21.html   (549 words)

Expression of Novel ING Variants Is Regulated by Thyroid Hormone in the Xenopus laevis Tadpole -- Wagner et al. 276 ... PCR was performed for all the RACE-PCR reactions (five cycles The PCR reactions were run on 1% agarose gels and transferred to Nytran Plus membrane (Mandel Scientific Co. Ltd., Mississauga, Those PCR reactions found to be positive for frog ING sequences through Southern blotting were used directly in cloning or, www.jbc.org /cgi/content/full/276/50/47013   (6197 words)

Failure to detect Chlamydia pneumoniae DNA in cerebral aneursymal sac tissue with two different polymerase chain ... Failure to demonstrate Chlamydia Pneumoniae in symptomatic abdominal aortic anuerysms by a nested polymerase chain reaction (PCR). Touchdown enzyme time release-PCR for detection and identification of Chlamydia trochomatis, C. pneumoniae, and C.psittaci using the 16S and 16S-23S spacer rRNA genes. Analytical sensitivity, reproducibility of results and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral mononuclear cells. jnnp.bmjjournals.com /cgi/content/full/74/6/756   (2256 words)

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