PCR & DNA Sequencing. PCR= Polymerase Chain Reaction. PCR applications

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "PCR & DNA Sequencing. PCR= Polymerase Chain Reaction. PCR applications"

Transcription

1 PCR= Polymerase Chain Reaction PCR & DNA Sequencing Biology 224 Instructor: Tom Peavy March 20, 2006 DNA photocopier integral tool for molecular biologists work horse versatile (many applications) not difficult to perform technically fast <Figures from PCR by McPherson & Moller> PCR applications Kary Mullis of Cetus Corp invented PCR in 1983 (Klenow fragment of DNA pol I) First paper describing the use of Taq polymerase was in 1988 (Saiki et al., 1988) PCR patent issues involving Taq polymerase Cloning cdnas and RAPD Cloning genes Real-time PCR PCR mutagenesis (*) PCR probe generation for hybridization Population sampling and genotyping Genomic fingerprinting RAPD-PCR p 253; multiplex-pcr p255 PCR-VNTRs p 257, Micro- and Minisatelline repeat-pcr p 260) Diagnostic PCR (detection of pathogens, GMOs, etc.)(*) 1

2 PCR components Template DNA Primers dntps (water, buffer) Thermostable polymerase 1) Template DNA is denatured (Denaturation phase; 94 C) 2) Primers allowed to anneal to template; Tm of primers is important (Annealing phase; variable temperature) 3) Increase temperature to optimum for thermostable polymerase (Elongation phase; C) 4) Repeat the whole cycle starting at step 1 PCR Kinetics Early cycles: primers act like probes searching for complementary Sequences on template DNA Mid cycles: amplication process is is fully underway with an exponential accumulation of amplimers Late cycles: reagents are limiting and Amplification is suboptimal 2

3 Genomic DNA Sources of Template DNA RNA isolation and cdna Plasmid, bacteriophage, cosmid and artifical chromosome DNA Pathological and forensic samples Archaeological samples Template amount -very sensitive technique (don t need much target template) but the amount of template is likely to require optimization (generally <1 nanogram of cloned template and up to a microgram of genomic DNA are used) -relative amount of target can be increased (e.g. choose cdna library where target should be expressed in large amounts) Technical Difficulties Mispriming primers anneal to alternate sites and not to correct or targeted site Needle-in-a-haystack (Template in limited amounts) Mismatches allowed internally if annealing temperature is low (below Tm) Misprimed PCR products will continue to be amplified (PCR primers are incorporated into the amplimer at the terminal end and will thus serve as a perfect match for future PCR cycles; large amounts of PCR product accumulate if in it occurs in the early cycles) 3

4 PCR of non-specific sequences or misprimed products leads to either smeary gels or unexpected amplimers sizes Artifactual products on agarose gels can arise from Primer-Dimer formation Contamination Problems Carry-over contamination prior PCR products, clones, or samples with DNA in general (e.g. cell lysates, genomic or plasmid DNA preparations, etc) can enter the PCR tube and serve as potential template -most often due to aerosol from pipetteman Other template contamination can be due to: - floating debris (circulation/vents) -laboratory surfaces -tissue from self or others (e.g. skin, hair) -solution contamination Preventative: aerosol-free tips (cotton plugs), UV irradiation, Designated PCR set-up area, gloves, premixes, aliquots of reagents Optimization of PCR To improve specificity: template quality optimize concentrations of Mg 2+, other ions, primers, dntps and polymerase (*) efficient denaturation, high annealing temps, and fast ramping rates limiting number of cycles and their length PCR strategies (e.g. touchdown PCR, hot start PCR nested PCR) Primer design (*) 4

5 Magnesium ions are critical -Magnesium ion concentration often needs optimization exists as dntp-mg 2+ complexes, interacts with DNA backbone influences activity of Taq polymerase; MgCl 2 is used in buffer to adjust concentrations To enhance for efficient denaturation, high annealing temps, and fast ramping rates: Use quality PCR machines (may not effectively reach temperatures or takes long time to ramp) Use thin walled PCR tubes Between 0.5 and 5mM is generally used (1.5 mm common) Low concentrations tend to have low yields of PCR product High concentrations tend to reduce the fidelity of Taq polymerase and lead to amplification of non-specific products NaCl or KCl concentrations also can be optimized Use Polymerase with High fidelity Taq polymerase (from thermophilic bacterium Thermus aquaticus) = 94 kda protein with 2 catalytic properties DNA polymerase (elongates nucleotides/second) exonuclease (removes nts in front of growing strand) Recombinant versions of Taq (enhanced for either purification or performance) Lacks 3 5 exonuclease activity Fidelity of Taq or error rate is: 1 base misincorporation per 10 4 nucleotides polymerized for a 400 bp fragment amplied 10 6 fold (=20 cycles) results in in about 33% of the products carrying a mutation (thus should sequence several PCR amplimers to determine consensus) 5

6 Proofreading DNA polymerases (= those that contain 3 5 exonuclease activity) Proofreading ability is due to the capacity of the enzyme to discriminate between whether the nucleotide at the 3 OH of an extending strand is correctly or incorrectly paired with the template strand Generally these enzymes are even more thermostable and tolerant of buffer conditions However, could chew up mismatched 3 primer ends also ( nibbling ) Increases fidelity about 5-12 depending on enzyme Examples: Vent, DeepVent, Tli (Thermoccocus litoralis), Pfu (Pyrococcus furiosus), 12 fold Hot Start PCR Used to overcome non-specific annealing of primers and/or primer-dimer formation prior to the denaturation step (annealed primers will be extended as temperature ramps up to denaturation temp) Cheapest method is to add polymerase after temperature is is above 70 C Alternatively can use commercial reagents such as Taq that has an antibody attached so as to prevent polymerase activity until the antibody is denatured (> 70 C) Usually leaves blunt ends for cloning rather than overhangs Touchdown PCR Used to increase specific PCR products Annealing temperature is set slightly above the Tm of the primers in the early cycles (enhances the chances of specific annealing of primers vs. non-specific) Annealing temperature is gradually lowered in subsequent cycles (e.g. 1 C every two cycles) until desired lower limiting annealing temperature is reached Nested PCR Design two outside primers for the first reaction, Then use a portion of the first reaction as template in a second reaction using Internal nested primers Effect is that the target sequences are preferentially amplified in early cycles and then are continued to be amplified exponentially (out competing non-specific targets) 6

7 Annealing Temperature and Primer Design Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a DNA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is: Tm = 4(G + C) + 2(A + T) o C In setting the annealing temperature of PCR reaction: As a rule of thumb, use an annealing temperature (Ta) about 5 o C below the lowest Tm of the pair of primers to be used if a good yield of product is desired Alternatively, if an increased specificity is desired, one can either Perform touchdown PCR (high-low anneal temp) The Tm of the two primers should not be different because it may never give appreciable yields of product due to trade-offs (annealing temperature appropriate for one but not the other) Can result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand. Note: Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long. Primer Length The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner (to avoid large differences) Another prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. Lengths are generally 17-25mers (rationale: there is a ¼ chance of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 4 16 bases (=4,294,967,296, or 4 billion): Primers can be designed with engineered sites at the 5 end (e.g. restriction enzyme sites, mutations) Mismatches can also be designed internally to facilitate in situ mutations (change coding sequence or create restriction sites) EcoRI Note: only use the annealing portion to calculate Tm 7

8 Degenerate Primers For amplification of sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: Degenerate primers= a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences. Need to examine all the options for particular amino acids with Respect to their codon degeneracy For the opposite direction (5 end race) need to reverse complement the sequence! 5 3 complement reverse 3 5 CGN CTG TGN CTT ACC CTG TTT CCN CTT GTG CCN A C A C C A 5 3 NCC GTG TTC NCC TTT GTC CCA TTC NGT GTC NGC A C C A C A 8

9 Design of degenerate primers based on amino acid sequencing: If you do not know where the peptide regions are located in the gene, then need to design PCR primers in both directions and try various combinations Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases Increased degeneracy means concentration of the individual primers decreases (of which there is only one exact match) thus, greater than 512-fold degeneracy should be avoided. 5 3 GTG TTC NCC TTT GTC CCA TTC NGT A C C A C (24mer) degeneracy= (1/4) 2 (1/2) 5 = 1/512 General Rules for Primer Design Can use deoxyinosine (di) at degenerate positions rather than use mixed oligos: di base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dis in an oligo - primers should be bases in length; - base composition should be 50-60% (G+C); - primers should end (3') in a G or C, or CG or GC (prevents "breathing" of ends and increases efficiency of priming) - Tms between o C are preferred; - runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided; - 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; - primer self-complementarity (ability to form 2 o structures such as hairpins) should be avoided. 9

10 Examples of inter- and intra-primer complementarity which would result in problems: Real-time PCR quantitation Multiplex PCR - uses multiple PCR primer sets to amplify Two or more products within single reaction - used for genotyping applications where simultaneous analysis of multiple markers is advantageous (or statistically necessary) - Can amplify over short tandem repeats (STRs) 10

11 Short Tandem Repeats (STRs) AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another 11

POLYMERASE CHAIN REACTION (PCR)

POLYMERASE CHAIN REACTION (PCR) POLYMERASE CHAIN REACTION (PCR) The polymerase chain reaction (PCR) is a technique used to amplify specific segments of DNA that may range in size from ca. 200-2000 or more base pairs. Two recent papers

More information

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) 05 PCR primer design 05.01 Design criteria PCR: an overview (from 02.01) PCR fidelity and optimization Primer design alessandro bogliolo isti information science and technology institute 1/20 Polymerase

More information

Lecture 37: Polymerase Chain Reaction

Lecture 37: Polymerase Chain Reaction Lecture 37: Polymerase Chain Reaction We have already studied basics of DNA/RNA structure and recombinant DNA technology in previous classes. Polymerase Chain Reaction (PCR) is another revolutionary method

More information

4 General PCR Methods Page

4 General PCR Methods Page Table of Contents General PCR Methods Page PCR Protocol Selection Guide...65.1 Basic PCR... 66.1.1 Hot Start PCR - The new Standard... 66.1.1.1 Reagents and Equipment Required... 66.1.1.2 General Considerations

More information

PCR-based technologies

PCR-based technologies Using molecular marker technology in studies on plant genetic diversity DNA-based technologies PCR-based technologies PCR basics Copyright: IPGRI and Cornell University, 2003 PCR basics 1 Contents! The

More information

BacReady TM Multiplex PCR System

BacReady TM Multiplex PCR System BacReady TM Multiplex PCR System Technical Manual No. 0191 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI Detailed Experimental

More information

PrimeSTAR HS DNA Polymerase

PrimeSTAR HS DNA Polymerase Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction

More information

Recombinant DNA & Genetic Engineering. Tools for Genetic Manipulation

Recombinant DNA & Genetic Engineering. Tools for Genetic Manipulation Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna

More information

PCR Optimization: Reaction Conditions and Components Sample Volume and Reaction Tubes Vapor Barrier and Thermal Transfer Fluid Template DNA or RNA

PCR Optimization: Reaction Conditions and Components Sample Volume and Reaction Tubes Vapor Barrier and Thermal Transfer Fluid Template DNA or RNA PCR Optimization: Reaction Conditions and Components The GeneAmp PCR process is widely employed in a tremendous variety of experimental applications to produce high yields of specific DNA target sequences.

More information

Extraction from Buccal Epithelial Cells

Extraction from Buccal Epithelial Cells Genomic DNA Extraction from Buccal Epithelial Cells The purpose of this lab is to collect a DNA sample from the cells that line the inside of your mouth and to use this sample to explore one of the most

More information

Blend Taq / Blend Taq -Plus-

Blend Taq / Blend Taq -Plus- Instruction manual Blend Taq and Blend Taq -Plus- 0810 F0938K Blend Taq / Blend Taq -Plus- BTQ-101 250 U 200 reactions BTQ-201 250 U 200 reactions Contents [1] Introduction

More information

HiPer RT-PCR Teaching Kit

HiPer RT-PCR Teaching Kit HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The

More information

BIOTECHNOLOGY. What can we do with DNA?

BIOTECHNOLOGY. What can we do with DNA? BIOTECHNOLOGY What can we do with DNA? Biotechnology Manipulation of biological organisms or their components for research and industrial purpose Usually manipulate DNA itself How to study individual gene?

More information

PrimeSTAR GXL DNA Polymerase

PrimeSTAR GXL DNA Polymerase For Research Use PrimeSTAR GXL DNA Polymerase Product Manual Table of Contents I. Description...3 II. Components...3 III. Storage...3 IV. Protocols...4 V. Optimization of Parameters...6 VI. Electrophoresis,

More information

PicoMaxx High Fidelity PCR System

PicoMaxx High Fidelity PCR System PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED

More information

SYBR Premix Ex Taq (Tli RNaseH Plus)

SYBR Premix Ex Taq (Tli RNaseH Plus) Cat. # RR420A For Research Use SYBR Premix Ex Taq (Tli RNaseH Plus) Product Manual Table of Contents I. Description... 3 II. Principle... 3 III. Components... 4 IV. Materials Required but not Provided...

More information

PCR: Polymerase Chain Reaction

PCR: Polymerase Chain Reaction PCR: Polymerase Chain Reaction Basics & Miniaturization Shared Nobel Prize - Chemistry 1993 for his invention of the polymerase chain reaction (PCR) method Outline What is PCR? DNA Background PCR Real

More information

PCR Polymerase Chain Reaction

PCR Polymerase Chain Reaction Biological Sciences Initiative HHMI PCR Polymerase Chain Reaction PCR is an extremely powerful technique used to amplify any specific piece of DNA of interest. The DNA of interest is selectively amplified

More information

GenScript BloodReady TM Multiplex PCR System

GenScript BloodReady TM Multiplex PCR System GenScript BloodReady TM Multiplex PCR System Technical Manual No. 0174 Version 20040915 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI

More information

Real-Time PCR Vs. Traditional PCR

Real-Time PCR Vs. Traditional PCR Real-Time PCR Vs. Traditional PCR Description This tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation. Objectives

More information

Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company

Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Biotechnology and reporter genes Here, a lentivirus is used to carry foreign DNA into chickens. A reporter gene (GFP)indicates that foreign DNA has been successfully transferred. Recombinant DNA continued

More information

Introduction To Real Time Quantitative PCR (qpcr)

Introduction To Real Time Quantitative PCR (qpcr) Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company www.sabiosciences.com The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors

More information

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. Instruction manual KOD -Plus- 0902 F0934K KOD -Plus- Contents [1] Introduction [2] Components [3] Quality testing [4] Primer design [5] Cloning of PCR products [6] Protocol 1. Standard reaction setup 2.

More information

PyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-)

PyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-) PyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608)

More information

TaKaRa Ex Taq 1PCR PRODUCTS. High Performance PCR. Kit Components RR001A. Applications RR01AM. Description. Concentration. Form. Storage.

TaKaRa Ex Taq 1PCR PRODUCTS. High Performance PCR. Kit Components RR001A. Applications RR01AM. Description. Concentration. Form. Storage. 1PCR PRODUCTS TaKaRa Ex Taq TaKaRa Ex Taq Cat.# RR001A 250 U TaKaRa Ex Taq Cat.# RR001B 1000 U (4 250 U TaKaRa Ex Taq Cat.# RR001C 3,000 U (12 250 U TaKaRa Ex Taq (Mg 2+ free Buffer Cat.# RR01AM 250 U

More information

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) PR037 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Polymerase Chain Reaction (PCR) Teacher s Guidebook (Cat. # BE 305) think proteins!

More information

Overview. KAPAHiFi HotStart is the engineered KAPAHiFi DNA Polymerase with an antibody-based hot start technology and improved buffer system.

Overview. KAPAHiFi HotStart is the engineered KAPAHiFi DNA Polymerase with an antibody-based hot start technology and improved buffer system. Overview KAPAHiFi HotStart is the engineered KAPAHiFi DNA Polymerase with an antibody-based hot start technology and improved buffer system. KAPAHiFi HotStart exhibits: World leading fidelity confirmed

More information

DNA CLONING: amplification of unique DNA molecules. In vivo-in different host cells

DNA CLONING: amplification of unique DNA molecules. In vivo-in different host cells DNA CLONING DNA CLONING: amplification of unique DNA molecules In vitro-pcr In vivo-in different host cells POLYMERASE CHAIN REACTION (PCR) PCR THE POLYMERASE CHAIN REACTION (PCR) PROVIDES AN EXTREMELY

More information

Genetics Faculty of Agriculture and Veterinary Medicine

Genetics Faculty of Agriculture and Veterinary Medicine Genetics 10201232 Faculty of Agriculture and Veterinary Medicine Instructor: Dr. Jihad Abdallah Topic 15:Recombinant DNA Technology 1 Recombinant DNA Technology Recombinant DNA Technology is the use of

More information

Next Generation Polymerase Chain Reaction

Next Generation Polymerase Chain Reaction Next Generation Polymerase Chain Reaction Developed by Nobel laureate Kary Mullis in the 1980s, Polymerase Chain Reaction (PCR) is a molecular technology that allows fast and in vitro. It has since become

More information

Chapter 10 Manipulating Genes

Chapter 10 Manipulating Genes How DNA Molecules Are Analyzed Chapter 10 Manipulating Genes Until the development of recombinant DNA techniques, crucial clues for understanding how cell works remained lock in the genome. Important advances

More information

Chap 4 Synthesis, Sequencing and Amplification of DNA

Chap 4 Synthesis, Sequencing and Amplification of DNA Chap 4 Synthesis, Sequencing and Amplification of DNA Why chemical synthesis of DNA? [1] 1. Used as a probe for DNA detection (Hybridization and Southern blotting) 2. Synthesized DNA for the assembly of

More information

Table of Contents. I. Description... 2. II. Kit Components... 2. III. Storage... 2. IV. 1st Strand cdna Synthesis Reaction... 3

Table of Contents. I. Description... 2. II. Kit Components... 2. III. Storage... 2. IV. 1st Strand cdna Synthesis Reaction... 3 Table of Contents I. Description... 2 II. Kit Components... 2 III. Storage... 2 IV. 1st Strand cdna Synthesis Reaction... 3 V. RT-PCR, Real-time RT-PCR... 4 VI. Application... 5 VII. Preparation of RNA

More information

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) Paul C Winter, Belfast City Hospital, Belfast, UK The polymerase chain reaction is a technique that allows DNA molecules of interest (usually gene sequences) to be copied

More information

MOLECULAR BIOLOGY OVERVIEW NUCLEIC ACIDS: THE BASICS

MOLECULAR BIOLOGY OVERVIEW NUCLEIC ACIDS: THE BASICS MOLECULAR BIOLOGY OVERVIEW NUCLEIC ACIDS: THE BASICS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu

More information

PicoMaxx High Fidelity PCR System

PicoMaxx High Fidelity PCR System PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision B Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED

More information

Our new breed is the center of attention.

Our new breed is the center of attention. Our new breed is the center of attention. PfuUltra II enzyme for highest fidelity enzyme for superior yield Our next generation of high fidelity Pfu-based fusion enzymes sets a new standard in high fidelity

More information

GUIDELINES FOR DESIGNING PRIMERS

GUIDELINES FOR DESIGNING PRIMERS Primer guidelines, Howard Judelson, 10.06, p. 1 GUIDELINES FOR DESIGNING PRIMERS Proper primer design is important for applications in PCR, DNA sequencing, and hybridization. Here are some tips to help

More information

RT-PCR: Two-Step Protocol

RT-PCR: Two-Step Protocol RT-PCR: Two-Step Protocol We will provide both one-step and two-step protocols for RT-PCR. We recommend the twostep protocol for this class. In the one-step protocol, the components of RT and PCR are mixed

More information

Chap 4 Synthesis, Sequencing and Amplification of DNA

Chap 4 Synthesis, Sequencing and Amplification of DNA Chap 4 Synthesis, Sequencing and Amplification of DNA Why chemical synthesis of DNA? [1] 1. Used as a probe for DNA detection (Hybridization and Southern blotting) 2. Synthesized DNA for the assembly of

More information

How many of you have checked out the web site on protein-dna interactions?

How many of you have checked out the web site on protein-dna interactions? How many of you have checked out the web site on protein-dna interactions? Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. Find and be ready to discuss

More information

Reference Dye N/A ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes)

Reference Dye N/A ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) Comparison of TaKaRa s and qrt-pcr Kits SYBR Premix Ex Taq (Tli RNase H+) Premix Ex Taq (Probe qpcr) SYBR Premix Ex Taq II (Tli RNase H+) Cat.# RR420A RR390A RR820A Reactions/Kit 200 (50 µl reaction size)

More information

Reagents for PCR. Expertise in Amplification

Reagents for PCR. Expertise in Amplification Reagents for PCR Expertise in Amplification Selection Guide Hot Start Enzymes for Specific Requirements PCR Application: Routine PCR Fast PCR Hot Start Multiplex/ Sequencing Wide Variety/ Easy Access High

More information

Computer Programs for PCR Primer Design and Analysis

Computer Programs for PCR Primer Design and Analysis PCR Primer Design 19 2 Computer Programs for PCR Primer Design and Analysis Bing-Yuan Chen, Harry W. Janes, and Steve Chen 1. Introduction 1.1. Core Parameters in Primer Design 1.1.1. T m, Primer Length,

More information

RT31-020 20 rxns. RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl

RT31-020 20 rxns. RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl Components RT31-020 20 rxns RT31-050 50 rxns RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl 2x RT Master Mix (2) 200 µl 2 x 250 µl 5 x 200 µl RNase H (E. coli) 20 µl 2 x 25 µl

More information

IMBB 2013. Genomic DNA purifica8on

IMBB 2013. Genomic DNA purifica8on IMBB 2013 Genomic DNA purifica8on Why purify DNA? The purpose of DNA purifica8on from the cell/8ssue is to ensure it performs well in subsequent downstream applica8ons, e.g. Polymerase Chain Reac8on (PCR),

More information

Upgrade to the gold standard for high-fidelity PCR

Upgrade to the gold standard for high-fidelity PCR 3 Thermo Scientific Phusion and Phire DNA Polymerases Accurate, fast and powerful Polymerases for better PCR Extremely accurate, fast and robust amplification Instant activation hot start technology Direct

More information

Taq98 Hot Start 2X Master Mix

Taq98 Hot Start 2X Master Mix Taq98 Hot Start 2X Master Mix Optimized for 98C Denaturation Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com

More information

11/19/2008. Gene analysis. Sequencing PCR. Northern-blot RT PCR. Western-blot Sequencing. in situ hybridization. Southern-blot

11/19/2008. Gene analysis. Sequencing PCR. Northern-blot RT PCR. Western-blot Sequencing. in situ hybridization. Southern-blot Recombinant technology Gene analysis Sequencing PCR RNA Northern-blot RT PCR Protein Western-blot Sequencing Southern-blot in situ hybridization in situ hybridization Function analysis Histochemical analysis

More information

Polymerase Chain Reaction (PCR) Practical Review Manzoor Ahmed Thokar, MD

Polymerase Chain Reaction (PCR) Practical Review Manzoor Ahmed Thokar, MD 61 Polymerase Chain Reaction (PCR) Practical Review Manzoor Ahmed Thokar, MD Introduction: The Controversial take off: When Mullis developed the Polymerase Chain Reaction (PCR) in 1983, he was working

More information

First Strand cdna Synthesis

First Strand cdna Synthesis 380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences

More information

LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR)

LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR) LAB 16 HUMAN DNA TYPING USING PCR LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR) STUDENT GUIDE GOAL The goal of this lab is to use polymerase chain reaction (PCR) to compare a human DNA

More information

SybGREEN qpcr Primers and Standards

SybGREEN qpcr Primers and Standards SybGREEN qpcr Primers and Standards Tools for RT-PCR Application Guide Table of Contents Package Contents and Storage Conditions... 1 Introduction... 3 SybGREEN qpcr Primer Pairs... 3 Gene Specific qpcr

More information

Cloning Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems

Cloning Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems Promega Notes Number 71, 1999, p. 10 Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems By Kimberly Knoche, Ph.D., and Dan Kephart, Ph.D. Promega Corporation Corresponding

More information

The Techniques of Molecular Biology: Forensic DNA Fingerprinting

The Techniques of Molecular Biology: Forensic DNA Fingerprinting Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins

More information

Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet

Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet PCR Pre-Lab (pg. 1-3) PCR Pre-Lab Answers (pg. 4-7) RNAi Pre-Lab (pg. 8) RNAi Pre-Lab Answers (pg. 9-10 Gel Electrophoresis Worksheet (pg.

More information

360 Master Mix. , and a supplementary 360 GC Enhancer.

360 Master Mix. , and a supplementary 360 GC Enhancer. Product Bulletin AmpliTaq Gold 360 Master Mix and 360 DNA Polymerase AmpliTaq Gold 360 Master Mix AmpliTaq Gold 360 DNA Polymerase 360 Coverage for a Full Range of Targets AmpliTaq Gold 360 Master Mix

More information

Optimizing Real Time PCR: A Focused Approach for Exceptional Real-Time qpcr Results

Optimizing Real Time PCR: A Focused Approach for Exceptional Real-Time qpcr Results Optimizing Real Time PCR: A Focused Approach for Exceptional Real-Time qpcr Results Michael Wakem M.Sc Bio-Rad Laboratories Canada Ltd (800) 268-0213 Ext 3360 michael_wakem@bio-rad.com Overview Fundamentals

More information

Upgrade to the gold standard for high-fidelity PCR

Upgrade to the gold standard for high-fidelity PCR 3 Thermo Scientific Phusion and Phire DNA s Accurate, fast and powerful s for better PCR Extremely accurate, fast and robust amplification Instant activation hot start technology Direct loading on gels

More information

The correct answer is c B. Answer b is incorrect. Type II enzymes recognize and cut a specific site, not at random sites.

The correct answer is c B. Answer b is incorrect. Type II enzymes recognize and cut a specific site, not at random sites. 1. A recombinant DNA molecules is one that is a. produced through the process of crossing over that occurs in meiosis b. constructed from DNA from different sources c. constructed from novel combinations

More information

Popular Kheti. Volume -2, Issue-3 (July-September), 2014 Available online at popularkheti.info ISSN:

Popular Kheti. Volume -2, Issue-3 (July-September), 2014 Available online at popularkheti.info ISSN: Kheti Volume -2, Issue-3 (July-September), 2014 Available online at www.popularkheti.info 2014 popularkheti.info ISSN: 2321-0001 Polymerase Chain Reaction: A Robust Technology for Crop Improvement Vivekanand

More information

Speed Matters - Fast ways from template to result

Speed Matters - Fast ways from template to result qpcr Symposium 2007 - Weihenstephan Speed Matters - Fast ways from template to result March 28, 2007 Dr. Thorsten Traeger Senior Scientist, Research and Development - 1 - Overview Ạgenda Fast PCR The Challenges

More information

CHAPTER 14 LECTURE NOTES: RECOMBINANT DNA TECHNOLOGY

CHAPTER 14 LECTURE NOTES: RECOMBINANT DNA TECHNOLOGY CHAPTER 14 LECTURE NOTES: RECOMBINANT DNA TECHNOLOGY I. General Info A. Landmarks in modern genetics 1. Rediscovery of Mendel s work 2. Chromosomal theory of inheritance 3. DNA as the genetic material

More information

RevertAid Premium First Strand cdna Synthesis Kit

RevertAid Premium First Strand cdna Synthesis Kit RevertAid Premium First Strand cdna Synthesis Kit #K1651, #K1652 CERTIFICATE OF ANALYSIS #K1651 Lot QUALITY CONTROL RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generated a prominent

More information

RNA-related Products

RNA-related Products RNA-related Products TranscriptME RNA kit: Ideal choice for obtaining high yields of full-length cdna for RT-qPCR assays Suitable for as low RNA amount as 10 pg p p Convenient, reliable and cost-effective

More information

Technical Note. Roche Applied Science. No. LC 18/2004. Assay Formats for Use in Real-Time PCR

Technical Note. Roche Applied Science. No. LC 18/2004. Assay Formats for Use in Real-Time PCR Roche Applied Science Technical Note No. LC 18/2004 Purpose of this Note Assay Formats for Use in Real-Time PCR The LightCycler Instrument uses several detection channels to monitor the amplification of

More information

RNA-related Products

RNA-related Products RNA-related Products TRANSCRIPTME RNA kit: Ideal choice for obtaining high yields of full-length cdna for RT-qPCR assays Suitable for as low RNA amount as 10 pg p p Convenient, reliable and cost-effective

More information

Recombinant DNA Technology

Recombinant DNA Technology PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology The Role of Recombinant DNA Technology in Biotechnology Biotechnology

More information

Technical Manual No. 0173 Update Date 10112010

Technical Manual No. 0173 Update Date 10112010 TissueDirect TM Multiplex PCR System Technical Manual No. 0173 Update Date 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 3 V Simplified Procedures. 3 VI Detailed

More information

THE POLYMERASE CHAIN REACTION (PCR)

THE POLYMERASE CHAIN REACTION (PCR) THE POLYMERASE CHAIN REACTION (PCR) OBTECTIVE: To amplify a region of ribosomal DNA from a small amount of genomic DNA. INTRODUCTION: The polymerase chain reaction (PCR) was developed in 1983 by Kary Mullis,

More information

Reverse Transcription System

Reverse Transcription System TECHNICAL BULLETIN Reverse Transcription System Instruc ons for use of Product A3500 Revised 1/14 TB099 Reverse Transcription System All technical literature is available on the Internet at: www.promega.com/protocols/

More information

Lecture 10. mrna: Transcription Translation Start Translation Stop Transcription Start (AUG) (UAG, UAA, or UGA) Terminator S-D Sequence

Lecture 10. mrna: Transcription Translation Start Translation Stop Transcription Start (AUG) (UAG, UAA, or UGA) Terminator S-D Sequence Lecture 10 Analysis of Gene Sequences Anatomy of a bacterial gene: Promoter Coding Sequence (no stop codons) mrna: Transcription Translation Start Translation Stop Transcription Start (AUG) (UAG, UAA,

More information

Essentials of Real Time PCR. About Sequence Detection Chemistries

Essentials of Real Time PCR. About Sequence Detection Chemistries Essentials of Real Time PCR About Real-Time PCR Assays Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected

More information

DNA and Forensic Science

DNA and Forensic Science DNA and Forensic Science Micah A. Luftig * Stephen Richey ** I. INTRODUCTION This paper represents a discussion of the fundamental principles of DNA technology as it applies to forensic testing. A brief

More information

CHAPTER 8 RECOMBINANT DNA and GENETIC ENGINEERING

CHAPTER 8 RECOMBINANT DNA and GENETIC ENGINEERING CHAPTER 8 RECOMBINANT DNA and GENETIC ENGINEERING Questions to be addressed: How are recombinant DNA molecules generated in vitro? How is recombinant DNA amplified? What analytical techniques are used

More information

CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1

CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1 CAP 5510-8 BIOINFORMATICS Su-Shing Chen CISE 10/5/2005 Su-Shing Chen, CISE 1 Genomic Mapping & Mapping Databases High resolution, genome-wide maps of DNA markers. Integrated maps, genome catalogs and comprehensive

More information

Thermo Scientific Advanced PCR Enzymes

Thermo Scientific Advanced PCR Enzymes Thermo Scientific Advanced PCR Enzymes Thermo Scientific Phusion High-Fidelity DNA Polymerase Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase Thermo Scientific Phire Hot Start II DNA

More information

Troubleshooting Sequencing Data

Troubleshooting Sequencing Data Troubleshooting Sequencing Data Troubleshooting Sequencing Data No recognizable sequence (see page 7-10) Insufficient Quantitate the DNA. Increase the amount of DNA in the sequencing reactions. See page

More information

Sequencing a Genome: Inside the Washington University Genome Sequencing Center. Activity Supplement Paper PCR (DNA Amplification)

Sequencing a Genome: Inside the Washington University Genome Sequencing Center. Activity Supplement Paper PCR (DNA Amplification) Sequencing a Genome: Inside the Washington University Genome Sequencing Center Activity Supplement (DNA Amplification) Project Outline The multimedia project Sequencing a Genome: Inside the Washington

More information

GENOTYPING ASSAYS AT ZIRC

GENOTYPING ASSAYS AT ZIRC GENOTYPING ASSAYS AT ZIRC A. READ THIS FIRST - DISCLAIMER Dear ZIRC user, We now provide detailed genotyping protocols for a number of zebrafish lines distributed by ZIRC. These protocols were developed

More information

GoTaq Amplification Maximum performance, flexibility, control and convenience

GoTaq Amplification Maximum performance, flexibility, control and convenience GoTaq Amplification Maximum performance, flexibility, control and convenience GoTaq products and formulations designed to improve your amplification needs Reverse Transcription GoScript Reverse Transcriptase

More information

Recombinant DNA Technology

Recombinant DNA Technology Recombinant DNA Technology Dates in the Development of Gene Cloning: 1965 - plasmids 1967 - ligase 1970 - restriction endonucleases 1972 - first experiments in gene splicing 1974 - worldwide moratorium

More information

3. comparison with proteins of known function

3. comparison with proteins of known function Lectures 26 and 27 recombinant DNA technology I. oal of genetics A. historically - easy to isolate total DNA - difficult to isolate individual gene B. recombinant DNA technology C. why get the gene? 1.

More information

Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR)

Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Goals of the lab: 1. To understand how DNA s chemical properties can be exploited for purification 2. To learn practical applications of DNA purification

More information

An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH

An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH DNA basic principles DNA carries the information of life on Earth DNA is present in all organisms (some viruses use RNA)

More information

Lecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology

Lecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A,

More information

CompleteⅡ 1st strand cdna Synthesis Kit

CompleteⅡ 1st strand cdna Synthesis Kit Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: www.greenmountainbio.com Tel: 800-942-1160 Sales: Sales@ greenmountainbio.com Support:

More information

Chapter 20: Biotechnology: DNA Technology & Genomics

Chapter 20: Biotechnology: DNA Technology & Genomics Biotechnology Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms?

More information

Troubleshooting for PCR and multiplex PCR

Troubleshooting for PCR and multiplex PCR Page 1 of 5 Page designed and maintained by Octavian Henegariu (Email: Tavi's Yale email or Tavi's Yahoo email). As I am currently pursuing a new junior faculty position, the Yale URL and email may change

More information

DNA: A Person s Ultimate Fingerprint

DNA: A Person s Ultimate Fingerprint A partnership between the UAB Center for Community Outreach Development and McWane Center DNA: A Person s Ultimate Fingerprint This project is supported by a Science Education Partnership Award (SEPA)

More information

June 09, 2009 Random Mutagenesis

June 09, 2009 Random Mutagenesis Why Mutagenesis? Analysis of protein function June 09, 2009 Random Mutagenesis Analysis of protein structure Protein engineering Analysis of structure-function relationship Analysis of the catalytic center

More information

1/12 Dideoxy DNA Sequencing

1/12 Dideoxy DNA Sequencing 1/12 Dideoxy DNA Sequencing Dideoxy DNA sequencing utilizes two steps: PCR (polymerase chain reaction) amplification of DNA using dideoxy nucleoside triphosphates (Figures 1 and 2)and denaturing polyacrylamide

More information

Site-directed mutagenesis

Site-directed mutagenesis Site-directed mutagenesis Adrian Suarez Covarrubias Modified from Sherry Mowbray s 1 Content DNA, PCR, oligonucleotides Reverse PCR mutagenesis Quick change mutagenesis Multi-change mutagenesis Oligonucleotide

More information

DNA Sequencing Handbook

DNA Sequencing Handbook Genomics Core 147 Biotechnology Building Ithaca, New York 14853-2703 Phone: (607) 254-4857; Fax (607) 254-4847 Web: http://cores.lifesciences.cornell.edu/brcinfo/ Email: DNA_Services@cornell.edu DNA Sequencing

More information

HWK 9. Model answer: Mix positive control DNA with ligation mix; no or very few transformants

HWK 9. Model answer: Mix positive control DNA with ligation mix; no or very few transformants HWK 9 You are trying to clone a gene form your favorite organism in E. coli using a small plasmid vector. You have obtained no transformants. How would you attempt to solve this problem? Rather than listing

More information

Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid

Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid Lina Jew Department of Microbiology & Immunology, University of

More information

Lecture 38: DNA Fingerprinting

Lecture 38: DNA Fingerprinting Lecture 38: DNA Fingerprinting (DNA technology) The most awesome and powerful tool acquired by man since the splitting of atoms - The Time Magazine (USA) Conventional fingerprint of an individual comes

More information

2. True or False? The sequence of nucleotides in the human genome is 90.9% identical from one person to the next. False (it s 99.

2. True or False? The sequence of nucleotides in the human genome is 90.9% identical from one person to the next. False (it s 99. 1. True or False? A typical chromosome can contain several hundred to several thousand genes, arranged in linear order along the DNA molecule present in the chromosome. True 2. True or False? The sequence

More information

Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction

Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Objectives Understand the process of DNA isolation Perform DNA isolation using cheek cells Use thermal cycler and Taq polymerase to perform DNA

More information

Data Analysis for Ion Torrent Sequencing

Data Analysis for Ion Torrent Sequencing IFU022 v140202 Research Use Only Instructions For Use Part III Data Analysis for Ion Torrent Sequencing MANUFACTURER: Multiplicom N.V. Galileilaan 18 2845 Niel Belgium Revision date: August 21, 2014 Page

More information