DOCSLIB.ORG

Sahara Hot Start PCR Master Mix

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Sahara Hot Start PCR Master Mix USAGE NOTES Little/no product detected: Assay Design – Endpoint PCR: • Reagent was omitted from or improperly • Amplicon length: amplicons up to 4000 bp added to the assay. Verify complete adherence may be amplified. to protocol steps. Further optimization • Primer concentration: 100 nM is suitable for most of PCR protocol may be required. assays. May be varied between 50 – 1000 nM. • Check melt temperature of primers and lower the annealing temperature if needed. Template Preparation: • Improper qPCR channel selection. • Compatible templates: cDNA, gDNA, and plasmid. Ensure channel assignment matches probe. 990 Richard Ave, Suite 110 No purification is required for use of cDNA Santa Clara, CA 95050 with the Sahara Hot Start PCR Master Mix. Inconsistent replicate amplifications: United States • Purified DNA is ideal as PCR inhibition is avoidable. • Pipetting error. Ensure proper technique. Inhibition may occur with crude preparations • Insufficient mixing during preparation. www.chaibio.com despite inhibition resistance by the mix, Ensure thorough mixing of original resulting in a higher limit of detection. reagents. Ensure PCR reaction contents [email protected] • A range of 50 ng – 2 pg is typical for large genomes are adequately mixed following setup. +1 (800) 642-4002 Toll-free (i.e. human, mouse). The range of input DNA for +1 (650) 779-5577 International smaller genomes is greater. Efficiency out of range (< 90% or > 110%) Sahara Hot Start • GC content: Sahara Hot Start PCR Master Mix is or unexpected R² value: compatible with GC content from 40 – 70%. • Pipetting or dilution error during assay setup. PCR Master Mix • qPCR interference due to bubbles. Cloning: • Suboptimal reaction conditions. Chai™ and Sahara™ are trademarks of • Template concentration: may be increased • Inappropriate instrument threshold setting. Catalog #: R02151 Chai Biotechnologies Inc. up to 1000 ng to generate maximum product. For research use only • 3’ adenine overhang: a 3’ dA overhang is generated Amplification of No Template Control (NTC): by the Taq polymerase, allowing amplified products • Reagent contamination by DNA template. Store at: to be ligated using TA cloning. Excessive primer dimer formation: -20 ºC long-term • Suboptimal primer concentration or design. 4 ºC for 6 months TROUBLESHOOTING Redesign assay using software to check for www.chaibio.com Inconsistent plasmid amplifications: Avoid repeated freeze/thaw cycles primer dimer formation across all assays. Protect from light 002111 Rev A • Supercoiling of targets. Linearize the plasmid. INTRODUCTION PROTOCOL Reaction Setup: Thermocycling Protocol – Two-Step: Sahara Hot Start PCR Master Mix is a 2X mix for Prepare Master Mix: • Determine total volume required for reactions + 10%. Recommended for Real-Time PCR with endpoint PCR or quantitative probe-based detection • Thaw completely at room temperature. • Prepare assay mix for all components melt temperatures ≥ 60 ºC. using singleplex Real-Time PCR. Aptamer-based Mix contents by inversion or pipetting, except template. STEP TEMP. TIME hot start technology, unparalleled thermal stability, or by gently vortexing for < 3 s. • Aliquot reaction mix into PCR tubes/plate. compatibility with targets from an expansive range • Centrifuge to collect the solution at the • Add DNA template to PCR tubes/plate. Initial Denaturation 95 ºC 1 min of organisms, and high amplification efficiency bottom of the tube. • Close tubes/seal plate. Denature 95 ºC 15 s make it an ideal mix for routine PCR applications. • Centrifuge briefly Reaction Composition – Endpoint PCR: Extend 60 ºC 30 s Cycle 30 - 45x HIGHLIGHTS COMPONENT 20 μL REACTION FINAL CONC. Thermocycling Protocol – Three-Step: • Retains stability even under extreme conditions: Sahara Hot Start 10 μL 1X Recommended for endpoint PCR, or Real-Time PCR three months at 25 °C or eight days at 50 °C, USAGE NOTES PCR Master Mix with melt temperatures < 60 ºC. followed by 20 freeze/thaw cycles Assay Design – Real-Time PCR: • Aptamer-based hot start system reduces Forward & Reverse Primers variable 50 – 1000 nM STEP TEMP. TIME • Amplicon length: amplicon lengths of 80 – 200 bp produce optimal reaction efficiency values. non-specific amplification and primer dimers Template DNA variable < 1000 ng Initial Denaturation 95 ºC 1 min • Annealing temperature: a 60 °C annealing • Amplifies up to four kb from genomic DNA, Nuclease-free Water to 20 μL Denature 95 ºC 15 s and GC-rich targets with up to 70% GC-content and extension temperature is recommended for NOTE: Based on 20 μL reaction, adjust accordingly for other volumes. Anneal 45 – 68 ºC 15 – 30 s two-step Real-Time PCR. Perform three-step SPECIFICATIONS PCR if the T of the primer is < 60 °C. Extend 72 ºC 1 kb/min m Reaction Composition – Real-Time PCR: • Primer concentration: 400 nM is suitable for Polymerase Taq DNA Polymerase Final Extension 72 ºC 5 min most assays. Primer concentration should COMPONENT 20 μL REACTION FINAL CONC. GC-Rich Performance ≤ 70% Cycle 30 - 45x be varied to achieve optimum PCR assay Sahara Hot Start 10 μL 1X Max Amplicon Length 4 kb efficiencies (90 – 110%) for the target. PCR Master Mix • Probe concentrations: 250 nM is suitable for Hot Start Yes—Aptamer Forward & Reverse Primers variable 50 – 1000 nM most targets. For high abundance targets: Supported Probes TaqMan/Hydrolysis, Molecular Beacon, Probe variable 100 – 250 nM optimize the reaction using probe concentrations Scorpions ranging from 100 – 250 nM. Template DNA variable < 100 ng Supported Templates Genomic DNA, cDNA, Plasmid DNA Nuclease-free Water to 20 μL Continues on other side NOTE: Based on 20 μL reaction, adjust accordingly for other volumes..
Load more

Recommended publications
  • M0495datasheet-Lot0011212
    1X Standard Taq Reaction Buffer: Reaction setup: analyze primers. The final concentration of Hot Start 10 mM Tris-HCl Due to the hot start nature of the enzyme, reactions each primer in a PCR may be 0.05–1 µM, typi- 50 mM KCl can be assembled on the bench at room temperature cally 0.1–0.5 µM. Taq DNA Polymerase 1.5 mM MgCl 2 and transferred to a thermocycler. No separate activa- ++ pH 8.3 @ 25°C tion step is required to release the inhibitor from the 3. Mg and additives: 1-800-632-7799 enzyme. Mg++ concentration of 1.5–2.0 mM is optimal [email protected] Unit Definition: One unit is defined as the amount for most PCR products generated with Hot www.neb.com of enzyme that will incorporate 15 nmol of dNTP Add to a sterile thin-walled PCR tube: Start Taq DNA Polymerase. The final Mg++ M0495S 001121214121 into acid insoluble material in 30 minutes at 75°C. 25 µl 50 µl FInaL concentration in 1X Standard Taq Reaction COMPONENT REacTION REacTION CONCENTRATION Buffer is 1.5 mM. This supports satisfactory Unit Assay Conditions: 1X ThermoPol™ Reaction amplification of most amplicons. However, M0495S Buffer, 200 µM dNTPs including [3H]-dTTP and 10X Standard Taq Reaction Buffer 2.5 µl 5 µl 1X Mg++ can be further optimized in 0.5 or 1.0 mM 200 µg/ml activated Calf Thymus DNA. 200 units 5,000 U/ml Lot: 0011212 10 mM dNTPs 0.5 µl 1 µl 200 µM increments using MgCl2 (sold separately).
    [Show full text]
  • 11|1111 Technical Tips
    Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press 11|1111 Technical Tips PCR has revolutionized nucleic acid vapor barrier on the surface of the aque- Wax-embedded analysis in many scientific disciplines, ous mixture. With this approach, the PCR Reagents including molecular biology, medical di- wax must first be melted and solidified agnostics, population genetics, and fo- on top of the lower aqueous layer prior rensic analysis. (1~ PCR is a highly sensi- to addition of the missing compo- tive technique that can selectively nent(s). In a similar approach, wax is enrich for a specific target from a back- melted and solidified to cover the lower Patricia Blair, ground of nonrelated sequences. How- aqueous layer completely. (7~ The miss- Rama Ramanujam, and ever, if all of the reaction components ing reagent is then layered onto the wax Brent A. Burdick (nucleotides, buffer containing magne- cap, followed by two drops of mineral sium, primers, a thermostable DNA poly- oil. When the temperature of the tubes Pharmacia Biotech, Milwaukee, merase, and sample containing the tar- in the thermocycler exceeds the melting Wisconsin get to be amplified) are mixed at room temperature of the wax, the wax rises to temperature prior to thermocycling, pre- the top of the tube and the upper and mature mispriming events such as non- lower aqueous layers mix by convection. specific annealing of primers to nontar- Another variation of the hot start get nucleic acid sequences and the technique involves drying one of the re- production of primer oligomerization action components in trehalose and em- (primer dimer) artifacts may occur.
    [Show full text]
  • PCR Tools 2Nd Edition PCR Tools 2Nd Edition | 2009
    Novagen® PCR Tools 2nd edition PCR Tools 2nd edition | 2009 Thermostable DNA Polymerases ......... 4 Overview and Enzyme Selection Guide ................................. 4 KOD Hot Start DNA Polymerase ............................................. 6 KOD Hot Start Master Mix ....................................................... 8 KOD Xtreme™ Hot Start DNA Polymerase .............................. 9 KOD XL DNA Polymerase .......................................................11 KOD DNA Polymerase ............................................................12 NovaTaq™ Hot Start DNA Polymerase .................................13 NovaTaq Hot Start Master Mix Kit ........................................13 NovaTaq DNA Polymerase ....................................................14 NovaTaq PCR Master Mix .....................................................14 Taq Antibody ...........................................................................15 10 mM dNTP Mix ...................................................................15 Direct PCR from Blood ........................16 BloodDirect™ PCR Buffer Kits ...............................................16 RT-PCR ....................................................18 One Step RT-PCR Master Mix Kit ........................................18 First Strand cDNA Synthesis Kit ..........................................19 Cover and Inside Photography: Chris Bucher Photography, Dale Chihuly Sculpture located at Indiana University, Medical Sciences Building, Indiana, United States. Prices and availability
    [Show full text]
  • Pcr Hot Start Protocol Thermo Fisher
    Pcr Hot Start Protocol Thermo Fisher Lou is self-pleasing: she fortress imperceptibly and routings her degree. Ishmael still herd frolicsomely mishearwhile aetiological tremulously Riccardo when Silvanreorganise is unbid. that enterprise. Slant Putnam superintend capriciously or Screening for use any source of hot start pcr amplification Expression Assays Protocol 4334429 Primer Express Software Version 30 Getting. The fisher scientific experiments where the data collection of thermo fisher scientific taq is formulated for pcr hot start protocol thermo fisher scientific taq dna. Track dna polymerases with pcr may be easily organize all pcr hot start protocol thermo fisher scientific catalog number. New thermal insulation, thermo fisher scientific gains that starts with them unique in thermo fisher scientific protocol that mutation at every two replicates extracted dna sequencing to bind. Hot physically meaningful parameters can be performed, hot physically meaningful content for pcr hot start protocol thermo fisher scientific industries providing high yields compared using pcr test subject composition selected from. Cell biology lab equipment line, hot start pcr protocol times, hot start pcr applications; sharps in all primer which view the right or chemistry, the fidelity pcr. The thermo scientific direct loading yes, thermo fisher scientific. MyFuge Mini centrifuge shown includes rotors for microtube and PCR. It stick a growing in where thermo Fisher Scientific offers a sheet range. The thermo scientific protocol times due to pcr hot start protocol thermo fisher scientific laboratory safety supplies, muß das gerät unter normalen laborbedingungen verwendet wird. Axygen LightCycler 40 Compatible PCR Plates Thin walls coupled with the 40 platform. Jump dive to clutch is Hot-Start Technology Beneficial For Your PCR AU wwwthermofishercom.
    [Show full text]
  • Download Author Version (PDF)
    RSC Advances This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. This Accepted Manuscript will be replaced by the edited, formatted and paginated article as soon as this is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/advances Page 1 of 33 RSC Advances Employment of Nanomaterials in Polymerase Chain Reaction: Insight into the Impacts and Putative Operating Mechanisms of Nano-additives in PCR Meral Yuce, a* Hasan Kurt, b Venkata R.S.S. Mokkapati, a Hikmet Budak a, b a Sabanci University, Nanotechnology Research and Application Centre, 34956, Istanbul, Turkey b Sabanci University, Faculty of Engineering and Natural Sciences, 34956, Istanbul, Turkey Corresponding author: [email protected] Abstract Manuscript The unique ability to rapidly amplify low copy number DNA has made in vitro Polymerase Chain Reaction one of the most fundamental techniques in modern biology.
    [Show full text]
  • Genome Walking with 2- to 4-Kb Steps Using Panhandle PCR
    Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Genome Walking with 2- to 4-kb Steps Using Panhandle PCR Douglas H. Jones and Stanley C. Winistorfer Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa 52242-1083 Panhandle PCR is a method that per- The PCR permits the highly specific trastrand annealing, those single strands mits the rapid retrieval of DNA seg- amplification of DNA sequences, but re- of genomic DNA that contain the com- ments flanking a known DNA se- quires knowledge of the sequences that plement to the 3' ends wil! form a stem- quence. This method extends the flank both ends of the sequence of inter- loop structure with a recessed 3' end. application of PCR to the retrieval of est. ~1-3~ Several methods have been de- Therefore, the ligated oligonucleotide DNA where only one end of the DNA veloped to permit the PCR amplification can prime template-directed DNA poly- sequence is known, so that one can of unknown DNA that flanks one end of merization on those strands that contain use PCR to walk along an uncharac- a known sequence. (4-23) The number the known sequence, resulting in known terized stretch of DNA. In this report, and variety of methods developed to am- DNA being attached to the uncharacter- we demonstrate that this method can plify DNA flanking a known sequence at- ized end of the unknown DNA (Fig. 1, be applied to the highly specific re- test to the scientific need for an effective step 3).
    [Show full text]
  • Temperature-Sensitive DNA Aptamer Blocking Enzymatic Activity of Twa DNA Polymerase
    Temperature-sensitive DNA aptamer blocking enzymatic activity of Twa DNA polymerase Polymerase chain reaction (PCR) is a basic method in many fields of molecular biology and diagnosis, as well as an important tool for gene cloning and analysis. Taq DNA polymerase isolated from Thermus aquatics is the most widely used enzyme for PCR-based diagnosis. However, the enzyme does not have proofreading activity, and therefore is not suitable for gene cloning and amplification of long DNA fragments. To solve this problem, we recently isolated from Thermococcus waiotapuensis the enzyme Twa DNA polymerase, which has 3’-5’ exonuclease-dependent proofreading activity and is able to amplify longer DNA fragments than the Taq DNA polymerase. Although temperature-resistant DNA polymerases have maximum enzymatic activity at about 70oC, some polymerase activity is also observed at room temperature. Because at this temperature oligonucleotide primers could bind to the DNA template nonspecifically, unwanted nonspecific fragments are often generated when PCR is set up at room temperature. To prevent generation of the nonspecific fragments, hot-start PCR was developed in which the enzyme activity of DNA polymerase at temperature <40oC is blocked by chemical modification or by antibodies specific for the active site of the enzyme. Higher temperature attained during the first denaturation step of PCR (usually about 94oC) removes the inhibitory effect of the inhibitors. These strategies for hot-start PCR have drawbacks, because they reduce activity of the enzyme and/or are irreversible. An alternative method for hot-start PCR is to use a DNA aptamer which, similarly to antibodies, binds to the enzyme and inhibits its activity at low temperature.
    [Show full text]
  • Microdroplet-Based One-Step RT-PCR for Ultrahigh Throughput Single-Cell
    www.nature.com/scientificreports OPEN Microdroplet‑based one‑step RT‑PCR for ultrahigh throughput single‑cell multiplex gene expression analysis and rare cell detection Jennifer Ma1, Gary Tran2, Alwin M. D. Wan3, Edmond W. K. Young1,3, Eugenia Kumacheva1,3,4, Norman N. Iscove2,5,8 & Peter W. Zandstra6,7,8* Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single‑cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet‑ based, one‑step reverse‑transcriptase polymerase chain reaction (RT‑PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read‑out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate‑mediated inhibition and allows for one‑step RT‑PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single‑cell gene signature profles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single‑cell analysis and enable a wide range of research and clinical applications.
    [Show full text]
  • Detection of Antibiotic Resistance in Swine Production
    Detection of Antibiotic Resistance in Swine Production By: SEPIDEH PAKPOUR Department of Food Science and Agricultural Chemistry Macdonald Campus, McGill University Montreal, Quebec A thesis submitted to the Office of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the degree of Master of Science. August, 2010 © SEPIDEH PAKPOUR This thesis is dedicated to my first and beloved teachers: My parents Mr. Mohammad Reza Pakpour and Mrs. Esmat Abolmasomi. TABLE OF CONTENT TABLE OF CONTENT I LIST OF TABLES VII LIST OF FIGURES VIII LIST OF ABBREVIATIONS IX ABSTRACT X RÉSUMÉ XI ACKNOWLEDGMENTS XIII INTRODUCTION XV Hypotheses XVI Objectives XVII Experimental Approach XVII CONTRIBUTION TO RESEARCH XIX LIST OF PUBLICATIONS AND PRESENTATIONS XX CHAPTER 1. LITERATURE REVIEW 1 1.1 General Introduction to Antibiotics 1 1.1.1 Definition 1 1.1.2 History of Antibiotics 1 1.1.3 Classifications 2 1.1.4 How do Antibiotics Work? 2 1.1.5 Applications of Antibiotics 3 1.2 General Introduction to Antibiotic Resistance 4 I 1.2.1 Definition 5 1.2.2 Classification 6 1.2.2.1 Innate (Intrinsic) Resistance 6 1.2.2.2 Acquired (Extrinsic) Resistance 6 1.2.2.3 Adaptation 7 1.3 General Introduction to Swine Rearing 9 1.4 Antibiotic Resistance in Swine Production 11 1.5 Tetracyclines 12 1.5.1 Discovery and Development of Tetracyclines 12 1.5.2 Structure of Tetracyclines 14 1.5.3 Mode of Action 14 1.5.4 Resistance to Tetracyclines 17 1.5.4.1 Protection of the Ribosome 17 1.5.4.2 Efflux Proteins 19 1.5.4.3 Enzymatic Inactivation 19 1.5.4.4 Other/Unknown Mechanisms of Resistance 21 1.6 Macrolides 21 1.6.1 Discovery and Development of Macrolides 21 1.6.2 Structure of Macrolides 23 1.6.3 Mechanisms of Action of Macrolides 23 1.6.4 Resistance to Macrolides 23 1.6.4.1 Ribosomal Methylation 23 1.6.4.2 Efflux Systems 25 II 1.6.4.3 Enzymatic Inactivation 25 1.6.4.4 Mutational Resistance to Macrolides 25 1.7 General Introduction to Tylosin 26 1.8 Food-Borne Pathogens 27 CONNECTING STATEMENT TO CHAPTER 2 29 CHAPTER 2.
    [Show full text]
  • Datasheet for Hot Start Taq DNA Polymerase
    1X Standard Taq Reaction Buffer: Reaction setup: analyze primers. The final concentration of Hot Start 10 mM Tris-HCl Due to the hot start nature of the enzyme, reactions each primer in a PCR may be 0.05–1 µM, typi- 50 mM KCl can be assembled on the bench at room temperature cally 0.1–0.5 µM. Taq DNA Polymerase 1.5 mM MgCl 2 and transferred to a thermocycler. No separate activa- ++ pH 8.3 @ 25°C tion step is required to release the inhibitor from the 3. Mg and additives: 1-800-632-7799 enzyme. Mg++ concentration of 1.5–2.0 mM is optimal [email protected] Unit Definition: One unit is defined as the amount for most PCR products generated with Hot www.neb.com of enzyme that will incorporate 15 nmol of dNTP Add to a sterile thin-walled PCR tube: Start Taq DNA Polymerase. The final Mg++ M0495S 001120714071 into acid insoluble material in 30 minutes at 75°C. 25 µl 50 µl FINAL concentration in 1X Standard Taq Reaction COMPONENT REACTION REACTION CONCENTRATION Buffer is 1.5 mM. This supports satisfactory Unit Assay Conditions: 1X ThermoPol™ Reaction 10X Standard Taq amplification of most amplicons. However, M0495S Buffer, 200 µM dNTPs including [3H]-dTTP and Reaction Buffer 2.5 µl 5 µl 1X Mg++ can be further optimized in 0.5 or 1.0 mM 200 µg/ml activated Calf Thymus DNA. 200 units 5,000 U/ml Lot: 0011207 10 mM dNTPs 0.5 µl 1 µl 200 µM increments using MgCl2 (sold separately).
    [Show full text]
  • Nucleic Acid Amplification Techniques
    ACTA SCIENTIFIC MEDICAL SCIENCES Volume 2 Issue 9 December 2018 Review Article Mukul Dev1,3, ShitanshuNucleic Kaundal Acid 2Amplification, Narotam Sharma Techniques3*, Satish Chandra(NAAT) Nautiyalin Post 3Genomics, Bhageshwari Era Mahato3, Preeti3, Manish Dev Sharma4 and Gulshan Kumar Dhingra5 1Krishna Adarsh College of Education Bhotwas, Rewari, Haryana, India 2Department of Microbiology, Shri Dev Suman Subharti Medical College, Ras Bihari Bose Subharti University, Dehradun, Uttrakhand, India 3Central Molecular Research Laboratory, Biochemistry Department, SGRRIM&HS, Shri Guru Ram Rai University, Patel Nagar, Dehradun, Uttarakhand, India 4College of Basic and Applied Sciences, Shri Guru Ram Rai University, Dehradun, Uttarakhand, India 5Pt. L. M. S. Govt. P.G. College, Rishikesh, Uttarakhand, India *Corresponding Author: Narotam Sharma, Scientist, Central Molecular Research Laboratory, Biochemistry Department, SGRRIM&HS, ShriReceived: Guru Ram Rai University,Published: Patel Nagar, Dehradun, Uttarakhand, India. October 22, 2018; November 19, 2018 Abstract - Nucleic Acid Amplification Techniques (NAAT) have evolved quickly with new innovations which can provide unprecedented op portunities in the field of Health and food security. Over time different method of Nucleic Acid Extraction (NAE) has been evolved like from a chemical based or solution based method to solid based to magnetic beads bases method and now we are in the period of full automation, we got fully automated Nucleic Acid (NA) extractors, a chip based system for amplification which take much less time in comparison to other amplification systems. More evolution in amplification techniques with introduction of Helicase Dependent - DNA Amplification (HDA) which is superior to all other methods in terms of ease of performance, cost, and its ability to perform a completely isothermal reaction.
    [Show full text]
  • Overview of Thermostable DNA Polymerases for Classical PCR Applications: from Molecular and Biochemical Fundamentals to Commercial Systems
    Appl Microbiol Biotechnol (2013) 97:10243–10254 DOI 10.1007/s00253-013-5290-2 MINI-REVIEW Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems Kay Terpe Received: 22 July 2013 /Revised: 20 September 2013 /Accepted: 22 September 2013 /Published online: 1 November 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract During the genomics era, the use of thermostable before becoming standard. Time and temperature of denaturing DNA polymerases increased greatly. Many were identified and can deactivate the polymerase more or less depending on the described—mainly of the genera Thermus, Thermococcus and used enzyme. Factors like DNA origin, primer and product Pyrococcus. Each polymerase has different features, resulting length as well as guanine–cytosine content should have a direct from origin and genetic modification. However, the rational influence on the choice of polymerase (Wu et al. 1991). Salt, choice of the adequate polymerase depends on the application magnesium and deoxyribonucleotide triphosphate (dNTP) con- itself. This review gives an overview of the most commonly centrations can greatly affect the PCR (Ling et al. 1991; used DNA polymerases used for PCR application: KOD, Pab Owczarzy et al. 2008). Additives like BSA (Al-Soud and (Isis™), Pfu, Pst (Deep Vent™), Pwo, Taq, Tbr, Tca, Tfi, Tfl, Rådström 2001), dimethylsulfoxide (Chester and Marshak Tfu, Tgo, Tli (Vent™), Tma (UITma™), Tne, Tth and others. 1993), formamide (Sarkar et al. 1990), betaine (Henke et al. 1997; Rees et al. 1993), ethylene glycol and 1,2-propanediol Keywords Thermostable DNA polymerase . Polymerase (Zhang et al. 2009), and others (Al-Soud and Rådström 2000; chain reaction (PCR) .
    [Show full text]