The GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity

Save this PDF as:
Size: px
Start display at page:

Download "The GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity"

Transcription

1 Promega Notes Magazine Number 62, 1997, p. 02 The GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity By Christine Andrews and Scott Lesley Promega Corporation Promega's new GeneEditor TM in vitro Site-Directed Mutagenesis System * provides a high efficiency, reliable procedure for the generation and selection of oligonucleotide-directed mutants. The Selection s provided in this system encode mutations which alter the substrate specificity of beta-lactamase, the gene encoding ampicillin resistance, commonly found on cloning vectors. This altered substrate specificity provides a positive selection for the mutant strand and results in consistently high mutagenesis frequencies using either double-stranded (ds) or single-stranded (ss) DNA templates. * Patent Pending. Introduction Site-directed mutagenesis is a valuable tool for the study of DNA function and protein structure and function. A number of different mutagenesis methods have been reported (1,2). Site-directed in vitro mutagenesis (3) is accomplished by hybridizing a target DNA to a synthetic oligonucleotide which is complementary to the target template except for a region of mismatch near the center. It is this region that contains the desired mutation. Following hybridization, the oligonucleotide is extended with DNA polymerase to create a doublestranded structure. The nick is then sealed with DNA ligase and the duplex structure is transformed into an E. coli host. The GeneEditor TM in vitro Site-Directed Mutagenesis System uses antibiotic resistance to select for plasmid derived from the mutant strand. This positive selection results in consistently high mutagenesis efficiencies (often >90%). If no selection method for mutants is employed, the theoretical yield of mutants using this procedure is 50% (due to the semi-conservative mode of DNA replication). In practice, however, the mutant yield in the absence of selection may be much lower, often only a few percent or less. This is assumed to be due to such factors as incomplete in vitro polymerization, primer displacement by the DNA polymerase used in the fill-in reaction, and in vivo host-directed mismatch repair mechanisms which favor repair of the newly synthesized unmethylated DNA strand (4). Positive selection of the mutant strand The GeneEditor TM System (Figure 1) alters ampicillin resistance to provide a selection which is specific to the newly synthesized mutant strand. Beta-lactam antibiotics, such as ampicillin, cause cell death in E. coli by inhibiting cell wall formation. Beta-lactamases confer resistance to ampicillin and other beta-lactam antibiotics (i.e., penicillins and cephalosporins) by degrading these compounds to an inactive form. The beta-lactamase enzyme inactivates these antibiotics by hydrolyzing the beta-lactam bond common to all penicillin and cephalosporin derivatives. These two broad classes of antibiotics contain many variations to the basic structure shown in Figure 2. The ability of beta-lactamase enzymes to recognize and degrade a variety of beta-lactam antibiotics has been well characterized (5). The wild-type TEM-1 beta-lactamase found on most cloning vectors is proficient at degrading antibiotics such as ampicillin. Cells expressing this beta-lactamase are therefore resistant to ampicillin. This resistance is used as a means of selection for the maintenance of most common cloning vectors. The TEM-1 enzyme is much less proficient at degrading some other beta-lactams, and cells containing the wild-type enzyme are sensitive to moderate levels of these antibiotics.

2 Figure 1. Schematic diagram of the GeneEditor TM in vitro Site-Directed Mutagenesis procedure. Figure 2. Structure of beta-lactam antibiotics. Numerous mutations which alter the substrate specificity of the beta-lactamase enzyme have been identified. Cells harboring these mutations exhibit increased resistance to a broader spectrum of beta-lactam antibiotics while retaining resistance to ampicillin (6). These mutations provide a means to select for the mutant strand and are the basis for the high level of efficiency of the GeneEditor TM System. The GeneEditor TM in vitro Site-Directed Mutagenesis System The GeneEditor TM in vitro Site-Directed Mutagenesis System may be used with any vector containing an ampicillin resistance gene and the gene of interest. Figure 1 outlines the mutagenesis procedure, which can be performed using either dsdna or ssdna templates. A Selection provided with the system is annealed to the DNA template at the same time as the mutagenic oligonucleotide. Both the Selection and the mutagenic oligonucleotide must hybridize to the same strand and are used to prime synthesis of the mutant strand. Subsequent synthesis and ligation of this mutant strand links the two oligonucleotides. The mutagenesis reaction is transformed initially into a repair minus (muts) E. coli strain (BMH 71-18) to avoid repair of the desired mutation. The muts mutation prevents the formation of a repair complex at the point of the mismatch between the wild-type and mutant strand. The GeneEditor TM Site-Directed Mutagenesis System uses a proprietary mixture of antibiotics (the GeneEditor TM Antibiotic Selection Mix) to select only those cells containing the desired mutation. A subsequent strain transfer into E. coli JM109 and growth in media containing the GeneEditor TM Antibiotic Selection Mix ensures proper segregation of mutant and wild-type plasmids and results

3 in a high proportion of mutants. Mutants can be maintained using ampicillin alone for subsequent procedures. The GeneEditor TM System provides BMH and JM109 Competent Cells and the GeneEditor TM Antibiotic Selection Mix, as well as the required enzymes, buffers, and Selection s. A variety of bacterial strains were tested for sensitivity to the antibiotics used for selection. All of the strains used, including BMH (muts), ES1301 (muts), JM109, HB101, DH5alpha (Life Technologies, Inc.), XL1-Blue and SURE (Stratagene) showed the appropriate increase in resistance to the antibiotic when transformed with mutant DNA generated using the GeneEditor TM System. Making difficult mutations using the GeneEditor TM System Single base changes and other small mutations are the most common types of mutations created using conventional site-directed mutagenesis techniques. The generation of large insertions or deletions is often restricted by the intrinsic properties of the oligonucleotide primer and the target sequence. Base composition and formation of secondary structure in large primers can influence efficient hybridization to the target, and optimization of annealing conditions is often required. Suggestions for mutagenic oligonucleotide design and locations of additional resources are provided in reference 7. Positive selection of mutants with the GeneEditor TM System provides advantages over other site-directed mutagenesis systems. The antibiotic selection is efficient, simple to use and requires little DNA manipulation or hands-on time. The system is compatible with most popular cloning vectors and no subcloning is required. There also is no requirement for unique restriction sites or reliance on the efficiency of the restriction enzyme. Double-stranded template DNA can be used and there is no need to grow the template in a specialized bacterial strain. The T4 DNA Polymerase supplied with the system has high fidelity of incorporation and shows no strand displacement. This results in efficient incorporation of mutagenic oligonucleotides and less risk of undesirable secondary mutations than methods using other DNA polymerases. Various oligonucleotides were designed to assess the mutagenesis efficiencies of different types of mutations, including large insertions and deletions, using the GeneEditor TM System (Table 1). For large insertions and deletions, oligonucleotides were designed to include 20 perfectly matched base pairs on either side of the mutagenic region. To compare efficiencies using different types of DNA template, the GeneEditor TM System in vitro mutagenesis procedure was performed using both dsdna and ssdna derived from several different vectors. All the plasmid templates that were used contain the TEM-1 beta-lactamase gene. Mutagenic oligonucleotides were hybridized at a 25:1 oligonucleotide:template ratio, cooling from C at 1.5 C per minute using a thermal cycler. The percent efficiency of mutagenesis reactions for both dsdna and ssdna templates using each of these oligonucleotides and the GeneEditor TM System are shown in Table 1. Table 1. Mutations Generated Using the Gene Editor TM System. Mutagenic Vector Size Type of Mutation A pgem ** -3Zf(+) 32mer 4 base pair deletion in LacZ alphapeptide Efficiency dsdna template (%) 83% Efficiency ssdna template (%) B pgem -3Zf(+) 90mer 50 base pair insertion 59% 78% C pgem -3Zf (+) 40mer 50 base pair deletion 96% 87% D pgem -3Zf(+) 40mer 700 base pair deletion 64% 48% E pbr322 27mer 4 base pair deletion in Tet r gene % G pgem -7Zf(+) 5 oligos oligos encoding deletions and substitutions F pgem -11Zf(+) 23mer 4 base pair deletion in LacZ alphapeptide 73-75% 30% contain all 5 mutations H pkk223 41mer substitution encoding 2 amino acids 80% I pgem -4Z 42mer multiple substitutions % Clones were screened by blue/white screening, antibiotic resistance or by restriction digestion and were confirmed by size. to template molar

4 ratios were 25:1 as recommended in the GeneEditor TM in vitro Site-Directed Mutagenesis System Technical Manual #TM047. Hybridization conditions were 75 C for 5 minutes, followed by cooling to 37 C at a rate of 1.5 C per minute in a thermal cycler. **U.S. Pat. No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted. Results Mutation rates between 48% and 96% were obtained for all the single mutations created using the GeneEditor TM System. Comparable efficiencies were achieved using dsdna and ssdna templates. Thus, for most mutations, preparation of ssdna is not necessary. The results shown in Table 2 demonstrate linkage of the Selection with the mutagenic oligonucleotide. In this experiment, mutagenic oligonucleotides that induce a frameshift mutation in the LacZ alpha-peptide corresponding to the top or bottom strand were used with either the top or bottom strand Selection s using double-stranded pgem -11Zf(+) Vector DNA as a template. The results clearly show linkage of the selection and mutagenic oligonucleotides when hybridized to the same strand and demonstrate the power of the antibiotic selection in providing efficient mutagenesis. Mutagenic oligonucleotides can be used which hybridize to either DNA strand providing the correct Selection is used. Table 2. Linkage of Selection and Mutagenic s. Mutagenic Selection % Mutants bottom strand bottom strand 75 bottom strand top strand <1 top strand bottom strand <1 top strand top strand 73 Figure 3 shows the results of a typical mutant screen using restriction analysis. Mutants generated using oligonucleotide D (Table 1) were grown overnight in the presence of the GeneEditor TM Antibiotic Selection Mix. DNA from each clone was isolated and digested with Nde I and Pvu II. Digestion of the wild-type vector with these enzymes resulted in the generation of two bands of 580bp and 399bp (lane 3). The deletion of the 700bp target sequence in the mutant resulted in the production of a 279bp band (lanes 4, 6-8 and 10). In this example, 14 of a total of 22 colonies screened were positive for the deletion. In addition to single mutations, such as those shown in Figure 3, multiple mutations have also been created using the GeneEditor TM System. Multiple mutations can be generated by using more than one mutagenic oligonucleotide. We have introduced five separate mutagenic oligonucleotides encoding novel restriction sites into the pgem -7Zf(+) Vector with 30% efficiency using this system (Table 1.G). Figure 3. Nde I/Pvu II restriction digests on dsdna template, 700 base-pair deletion mutants. DNA from individual clones was prepared from 3ml cultures using Promega's Wizard Plus Minipreps DNA Purification System. Five microliters of each miniprep was incubated with 5 units each of Nde I and Pvu II at 37 C for 60 minutes. DNA fragments were separated on a 4-20% Tris-Glycine acrylamide gel and post-stained with ethidium bromide. Lanes: lane 1, pgem DNA Markers (Cat.# G1741); lane 2, uncut pgem -3Zf(+) Vector; lane 3, Nde I and Pvu II digest of wild-type pgem - 3Zf(+) Vector; lanes 4-10, Nde I and Pvu II digests of individual clone DNA. The deletion of a 700bp target sequence in the mutated DNA results in the production of a 279bp Nde I and Pvu II fragment. Nde I and Pvu II digestion of wild-type DNA results in the production of two fragments of 580bp and 399bp, respectively. Summary The GeneEditor TM in vitro Site-Directed Mutagenesis System offers several advantages over conventional approaches to oligonucleotide-directed mutagenesis. Like Promega's Altered-Sites II in vitro Mutagenesis System, the GeneEditor TM System relies on positive antibiotic selection of the mutant strand. This approach consistently results in a high proportion of mutants. Unlike other mutagenesis systems, the GeneEditor TM System can be used with most common cloning vectors that contain an ampicillin resistance gene, without subcloning into a specialized vector. The system uses the high fidelity T4 DNA Polymerase to reduce unwanted

5 secondary mutations. Difficult mutations can be generated using dsdna templates without extensive optimization of hybridization conditions. High mutagenesis efficiencies are consistently and easily achieved using the GeneEditor TM System. References 1. Smith, M. (1985) Ann. Rev. Genet. 19, Site-Specific Mutagenesis and Protein Engineering, Section IV, Chapters (1987) Meth. Enzymol. 154, Hutchinson, C.A. et al. (1978) J. Biol. Chem. 253, Kramer, B., Kramer, W. and Fritz, H.J. (1984) Cell 38, Matagne, A. et al. (1990) Biochem. J. 265, Venkatachalam, K.V. et al. (1994) J. Biol. Chem. 269, Cosby, N.C. and Lesley, S. (1997) Promega Notes 61, 12. Ordering Information Product GeneEditor TM in vitro Site-Directed Mutagenesis System Cat.# Q Promega Corporation. All Rights Reserved. Altered Sites, pgem and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. GeneEditor is a trademark of Promega Corporation. DH5alpha is a registered trademark of Life Technologies, Inc. SURE is a registered trademark of Stratagene.

3 Designing Primers for Site-Directed Mutagenesis

3 Designing Primers for Site-Directed Mutagenesis 3 Designing Primers for Site-Directed Mutagenesis 3.1 Learning Objectives During the next two labs you will learn the basics of site-directed mutagenesis: you will design primers for the mutants you designed

More information

Some types of Mutagenesis

Some types of Mutagenesis Mutagenesis What Is a Mutation? Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine

More information

Recitation CHAPTER 9 DNA Technologies

Recitation CHAPTER 9 DNA Technologies Recitation CHAPTER 9 DNA Technologies DNA Cloning: General Scheme A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown

More information

Chapter 15 Recombinant DNA and Genetic Engineering. Restriction Enzymes Function as Nature s Pinking Shears

Chapter 15 Recombinant DNA and Genetic Engineering. Restriction Enzymes Function as Nature s Pinking Shears Chapter 15 Recombinant DNA and Genetic Engineering In this chapter you will learn How restriction enzyme work and why they are essential to DNA technology. About various procedures such as cloning and

More information

Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene. Andrew ElBardissi, The Pennsylvania State University

Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene. Andrew ElBardissi, The Pennsylvania State University Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene Andrew ElBardissi, The Pennsylvania State University Abstract: Hong Ma, The Pennsylvania State University The Excess Microsporocytes

More information

BIOLOGY - CLUTCH CH.20 - BIOTECHNOLOGY.

BIOLOGY - CLUTCH CH.20 - BIOTECHNOLOGY. !! www.clutchprep.com CONCEPT: DNA CLONING DNA cloning is a technique that inserts a foreign gene into a living host to replicate the gene and produce gene products. Transformation the process by which

More information

Chapter 4. Recombinant DNA Technology

Chapter 4. Recombinant DNA Technology Chapter 4 Recombinant DNA Technology 5. Plasmid Cloning Vectors Plasmid Plasmids Self replicating Double-stranded Mostly circular DNA ( 500 kb) Linear : Streptomyces, Borrelia burgdorferi Replicon

More information

DNA Cloning with Cloning Vectors

DNA Cloning with Cloning Vectors Cloning Vectors A M I R A A. T. A L - H O S A R Y L E C T U R E R O F I N F E C T I O U S D I S E A S E S F A C U L T Y O F V E T. M E D I C I N E A S S I U T U N I V E R S I T Y - E G Y P T DNA Cloning

More information

Reading Lecture 3: 24-25, 45, Lecture 4: 66-71, Lecture 3. Vectors. Definition Properties Types. Transformation

Reading Lecture 3: 24-25, 45, Lecture 4: 66-71, Lecture 3. Vectors. Definition Properties Types. Transformation Lecture 3 Reading Lecture 3: 24-25, 45, 55-66 Lecture 4: 66-71, 75-79 Vectors Definition Properties Types Transformation 56 VECTORS- Definition Vectors are carriers of a DNA fragment of interest Insert

More information

Lecture Four. Molecular Approaches I: Nucleic Acids

Lecture Four. Molecular Approaches I: Nucleic Acids Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single

More information

Computational Biology I LSM5191

Computational Biology I LSM5191 Computational Biology I LSM5191 Lecture 5 Notes: Genetic manipulation & Molecular Biology techniques Broad Overview of: Enzymatic tools in Molecular Biology Gel electrophoresis Restriction mapping DNA

More information

7.1 Techniques for Producing and Analyzing DNA. SBI4U Ms. Ho-Lau

7.1 Techniques for Producing and Analyzing DNA. SBI4U Ms. Ho-Lau 7.1 Techniques for Producing and Analyzing DNA SBI4U Ms. Ho-Lau What is Biotechnology? From Merriam-Webster: the manipulation of living organisms or their components to produce useful usually commercial

More information

Molecular Genetics Techniques. BIT 220 Chapter 20

Molecular Genetics Techniques. BIT 220 Chapter 20 Molecular Genetics Techniques BIT 220 Chapter 20 What is Cloning? Recombinant DNA technologies 1. Producing Recombinant DNA molecule Incorporate gene of interest into plasmid (cloning vector) 2. Recombinant

More information

B. Incorrect! Ligation is also a necessary step for cloning.

B. Incorrect! Ligation is also a necessary step for cloning. Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease

More information

Amplified segment of DNA can be purified from bacteria in sufficient quantity and quality for :

Amplified segment of DNA can be purified from bacteria in sufficient quantity and quality for : Transformation Insertion of DNA of interest Amplification Amplified segment of DNA can be purified from bacteria in sufficient quantity and quality for : DNA Sequence. Understand relatedness of genes and

More information

Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19

Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19 Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19 IVANA KOMLJENOVIC Department of Microbiology and Immunology,

More information

Synthetic Biology for

Synthetic Biology for Synthetic Biology for Plasmids and DNA Digestion Plasmids Plasmids are small DNA molecules that are separate from chromosomal DNA They are most commonly found as double stranded, circular DNA Typical plasmids

More information

Site-directed Mutagenesis

Site-directed Mutagenesis Site-directed Mutagenesis Applications Subtilisin (Met à Ala mutation resistant to oxidation) Fluorescent proteins Protein structure-function Substrate trapping mutants Identify regulatory regions/sequences

More information

Step 1: Digest vector with Reason for Step 1. Step 2: Digest T4 genomic DNA with Reason for Step 2: Step 3: Reason for Step 3:

Step 1: Digest vector with Reason for Step 1. Step 2: Digest T4 genomic DNA with Reason for Step 2: Step 3: Reason for Step 3: Biol/Chem 475 Spring 2007 Study Problems for Quiz 2 Quiz 2 (~50 pts) is scheduled for Monday May 14 It will cover all handouts and lab exercises to date except the handout/worksheet (yet to be distributed)

More information

Regulation of enzyme synthesis

Regulation of enzyme synthesis Regulation of enzyme synthesis The lac operon is an example of an inducible operon - it is normally off, but when a molecule called an inducer is present, the operon turns on. The trp operon is an example

More information

Bootcamp: Molecular Biology Techniques and Interpretation

Bootcamp: Molecular Biology Techniques and Interpretation Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing

More information

Basics of Recombinant DNA Technology Biochemistry 302. March 5, 2004 Bob Kelm

Basics of Recombinant DNA Technology Biochemistry 302. March 5, 2004 Bob Kelm Basics of Recombinant DNA Technology Biochemistry 302 March 5, 2004 Bob Kelm Applications of recombinant DNA technology Mapping and identifying genes (DNA cloning) Propagating genes (DNA subcloning) Modifying

More information

Recombinant DNA Technology

Recombinant DNA Technology Recombinant DNA Technology Common General Cloning Strategy Target DNA from donor organism extracted, cut with restriction endonuclease and ligated into a cloning vector cut with compatible restriction

More information

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA)

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA) Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 6: Ligation & Bacterial Transformation (Bring your text and laptop to class if you wish to work on your assignment during

More information

Chapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering

Chapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering Chapter 8 Recombinant DNA and Genetic Engineering Genetic manipulation Ways this technology touches us Criminal justice The Justice Project, started by law students to advocate for DNA testing of Death

More information

Determination of Exclusion Effect in Wild Type and Rop Deficient Mutated pbr322 Co-transformations

Determination of Exclusion Effect in Wild Type and Rop Deficient Mutated pbr322 Co-transformations Determination of Exclusion Effect in Wild Type and Rop Deficient Mutated pbr322 Co-transformations Suzana Sabaiduc and Andy Lo Microbiology and Immunology University of British Columbia pbr322 is an expression

More information

Learning Objectives :

Learning Objectives : Learning Objectives : Understand the basic differences between genomic and cdna libraries Understand how genomic libraries are constructed Understand the purpose for having overlapping DNA fragments in

More information

In vitro mutagenesis

In vitro mutagenesis Core course BMS361N Genetic Engineering In vitro mutagenesis Prof. Narkunaraja Shanmugam Dept. Of Biomedical Science School of Basic Medical Sciences Bharathidasan University In vitro mutagenesis Many

More information

ET - Recombination. Introduction

ET - Recombination. Introduction GENERAL & APPLIED GENETICS Geert Van Haute August 2003 ET - Recombination Introduction Homologous recombination is of importance to a variety of cellular processes, including the maintenance of genomic

More information

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix Molecular Cloning Laboratories User Manual Version 3.3 Product name: Choo-Choo Cloning Kits Cat #: CCK-10, CCK-20, CCK-096, CCK-384 Description: Choo-Choo Cloning is a highly efficient directional PCR

More information

10X ligation buffer ligase 1 vector DNA insert DNA H 2 O. 10 µl Total Volume. 10X ligation buffer ligase 1 vector DNA insert DNA

10X ligation buffer ligase 1 vector DNA insert DNA H 2 O. 10 µl Total Volume. 10X ligation buffer ligase 1 vector DNA insert DNA Biol/Chem 475 S07 Study problems for quiz 1 See also questions posed in lab handouts including ligase handout Answers to questions 1&2 included at the end of this document. 1. You plan to clone a 1.0 kb

More information

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template Journal of Biomedicine and Biotechnology 2003:3 (2003) 202 207 PII. S1110724303209141 http://jbb.hindawi.com RESEARCH ARTICLE Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing

More information

Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase

Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase H. Liu, R. Ye and Y.Y. Wang Department of Medical Microbiology and Parasitology, School of

More information

BSCI410-Liu/Spring 06 Exam #1 Feb. 23, 06

BSCI410-Liu/Spring 06 Exam #1 Feb. 23, 06 Your Name: Your UID# 1. (20 points) Match following mutations with corresponding mutagens (X-RAY, Ds transposon excision, UV, EMS, Proflavin) a) Thymidine dimmers b) Breakage of DNA backbone c) Frameshift

More information

Technical tips Session 4

Technical tips Session 4 Technical tips Session 4 Biotinylation assay: Streptavidin is a small bacterial protein that binds with high affinity to the vitamin biotin. This streptavidin-biotin combination can be used to link molecules

More information

Genetic Engineering & Recombinant DNA

Genetic Engineering & Recombinant DNA Genetic Engineering & Recombinant DNA Chapter 10 Copyright The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Applications of Genetic Engineering Basic science vs. Applied

More information

Molecular Cell Biology - Problem Drill 11: Recombinant DNA

Molecular Cell Biology - Problem Drill 11: Recombinant DNA Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna

More information

Design. Construction. Characterization

Design. Construction. Characterization Design Construction Characterization DNA mrna (messenger) A C C transcription translation C A C protein His A T G C T A C G Plasmids replicon copy number incompatibility selection marker origin of replication

More information

Supplementary Information

Supplementary Information Supplementary Information Supplementary Figures Figure S1. Study of mgtl translation in vitro. (A) Detection of 5 LR RNA using wild-type and anti-sd (91-95) substituted templates in a transcription-translation

More information

p Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA

p Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA p.959-970 Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA - 2011-05 1 tetratricopeptide repeat Zona di possibile interazione con cargo BCMA - 2011-05 2 Verhey et

More information

Red Type Indicates Unique Site

Red Type Indicates Unique Site 3600 G0605 pscaavmcmvmcsbghpa Plasmid Features: Coordinates Feature 980-1084 AAV2 5 ITR 1144-1666 modified CMV 1667-1761 MCS 1762-1975 BgHpA 1987-2114 AAV2 3 ITR 3031-3891 B-lactamase (Ampicillin) Antibiotic

More information

Chapter 10 (Part II) Gene Isolation and Manipulation

Chapter 10 (Part II) Gene Isolation and Manipulation Biology 234 J. G. Doheny Chapter 10 (Part II) Gene Isolation and Manipulation Practice Questions: Answer the following questions with one or two sentences. 1. What does PCR stand for? 2. What does the

More information

Fun with DNA polymerase

Fun with DNA polymerase Fun with DNA polymerase Why would we want to be able to make copies of DNA? Can you think of a situation where you have only a small amount and would like more? Enzymatic DNA synthesis To use DNA polymerase

More information

Site-directed mutagenesis of proteins

Site-directed mutagenesis of proteins IFM/Kemi Linköpings Universitet August 2013/LGM Labmanual Site-directed mutagenesis of proteins Figur 1: Flow-chart of the site-directed mutagenesis lab exercise 2 Site-specific mutagenesis Introduction

More information

Bi 8 Lecture 4. Ellen Rothenberg 14 January Reading: from Alberts Ch. 8

Bi 8 Lecture 4. Ellen Rothenberg 14 January Reading: from Alberts Ch. 8 Bi 8 Lecture 4 DNA approaches: How we know what we know Ellen Rothenberg 14 January 2016 Reading: from Alberts Ch. 8 Central concept: DNA or RNA polymer length as an identifying feature RNA has intrinsically

More information

BACTERIAL GENETICS. How does the DNA in the bacterial cell replicate

BACTERIAL GENETICS. How does the DNA in the bacterial cell replicate BACTERIAL GENETICS Bacterial genetics is the study of gene structure and function in bacteria. Genetics itself is concerned with determining the number, location, and character of the genes of an organism.

More information

GenBuilder TM Plus Cloning Kit User Manual

GenBuilder TM Plus Cloning Kit User Manual GenBuilder TM Plus Cloning Kit User Manual Cat. No. L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2

More information

Overview: The DNA Toolbox

Overview: The DNA Toolbox Overview: The DNA Toolbox Sequencing of the genomes of more than 7,000 species was under way in 2010 DNA sequencing has depended on advances in technology, starting with making recombinant DNA In recombinant

More information

Ligation Independent Cloning (LIC) Procedure

Ligation Independent Cloning (LIC) Procedure Ligation Independent Cloning (LIC) Procedure Ligation Independent Cloning (LIC) LIC cloning allows insertion of DNA fragments without using restriction enzymes into specific vectors containing engineered

More information

Genetics and Genomics in Medicine Chapter 3. Questions & Answers

Genetics and Genomics in Medicine Chapter 3. Questions & Answers Genetics and Genomics in Medicine Chapter 3 Multiple Choice Questions Questions & Answers Question 3.1 Which of the following statements, if any, is false? a) Amplifying DNA means making many identical

More information

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES CHAPTER 11 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES POINTS TO REMEMBER Bacteriophage : A virus that infects bacteria. Bioreactor : A large vessel in which raw materials are biologically converted into

More information

Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 8: DNA Restriction Digest (II) and DNA Sequencing (I)

Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 8: DNA Restriction Digest (II) and DNA Sequencing (I) Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 8: DNA Restriction Digest (II) and DNA Sequencing (I) We have made considerable progress in our analysis of the gene for

More information

Chapter 8. Microbial Genetics. Lectures prepared by Christine L. Case. Copyright 2010 Pearson Education, Inc.

Chapter 8. Microbial Genetics. Lectures prepared by Christine L. Case. Copyright 2010 Pearson Education, Inc. Chapter 8 Microbial Genetics Lectures prepared by Christine L. Case Structure and Function of Genetic Material Learning Objectives 8-1 Define genetics, genome, chromosome, gene, genetic code, genotype,

More information

Chapter 9 Genetic Engineering

Chapter 9 Genetic Engineering Chapter 9 Genetic Engineering Biotechnology: use of microbes to make a protein product Recombinant DNA Technology: Insertion or modification of genes to produce desired proteins Genetic engineering: manipulation

More information

Molecular Biology Midterm Exam 2

Molecular Biology Midterm Exam 2 Molecular Biology Midterm Exam 2 1. The experiments by Frank Stahl and Matthew Messelson demonstrated that DNA strands separate during DNA replication. They showed that DNA replication is what kind of

More information

CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Section A: DNA Cloning

CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Section A: DNA Cloning Section A: DNA Cloning 1. DNA technology makes it possible to clone genes for basic research and commercial applications: an overview 2. Restriction enzymes are used to make recombinant DNA 3. Genes can

More information

The Biotechnology Toolbox

The Biotechnology Toolbox Chapter 15 The Biotechnology Toolbox Cutting and Pasting DNA Cutting DNA Restriction endonuclease or restriction enzymes Cellular protection mechanism for infected foreign DNA Recognition and cutting specific

More information

Your first goal is to determine the order of assembly of the 3 DNA polymerases during initiation.

Your first goal is to determine the order of assembly of the 3 DNA polymerases during initiation. MIT Department of Biology 7.28, Spring 2005 - Molecular Biology 1 Question 1 You are studying the mechanism of DNA polymerase loading at eukaryotic DNA replication origins. Unlike the situation in bacteria,

More information

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual Fusion Cloning technology Cold Fusion Cloning Kit Store the master mixture and positive controls at -20 C Store the competent cells at -80 C. (ver. 120909) A limited-use label license covers this product.

More information

GenBuilder TM Plus Cloning Kit User Manual

GenBuilder TM Plus Cloning Kit User Manual GenBuilder TM Plus Cloning Kit User Manual Cat.no L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2 Ⅱ.

More information

7.02 Recombinant DNA Methods Spring 2005 Exam Study Questions Answer Key

7.02 Recombinant DNA Methods Spring 2005 Exam Study Questions Answer Key MIT Department of Biology 7.02 Experimental Biology & Communication, Spring 2005 7.02/10.702 Spring 2005 RDM Exam Study Questions 7.02 Recombinant DNA Methods Spring 2005 Exam Study Questions Answer Key

More information

KOD -Plus- Mutagenesis Kit

KOD -Plus- Mutagenesis Kit Instruction manual KOD -Plus- Mutagenesis Kit 0811 F0936K KOD -Plus- Mutagenesis Kit SMK-101 20 reactions Store at -20 C Contents [1] Introduction [2] Flow chart [3] Components [4] Notes [5] Protocol 1.

More information

Biotechnology. Chapter 20. Biology Eighth Edition Neil Campbell and Jane Reece. PowerPoint Lecture Presentations for

Biotechnology. Chapter 20. Biology Eighth Edition Neil Campbell and Jane Reece. PowerPoint Lecture Presentations for Chapter 20 Biotechnology PowerPoint Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright

More information

Molecular Biology Techniques Supporting IBBE

Molecular Biology Techniques Supporting IBBE Molecular Biology Techniques Supporting IBBE Jared Cartwright Protein Production Lab Head Contact Details: email jared.cartwright@york.ac.uk Phone 01904 328797 Presentation Aims Gene synthesis Cloning

More information

Data Sheet Quick PCR Cloning Kit

Data Sheet Quick PCR Cloning Kit Data Sheet Quick PCR Cloning Kit 6044 Cornerstone Ct. West, Ste. E DESCRIPTION: The Quick PCR Cloning Kit is a simple and highly efficient method to insert any gene or DNA fragment into a vector, without

More information

Chapter 13: Biotechnology

Chapter 13: Biotechnology Chapter Review 1. Explain why the brewing of beer is considered to be biotechnology. The United Nations defines biotechnology as any technological application that uses biological system, living organism,

More information

Linköpings Universitet. Site-directed mutagenesis of proteins

Linköpings Universitet. Site-directed mutagenesis of proteins IFM/Kemi August2011/LGM Linköpings Universitet Site-directed mutagenesis of proteins Competent E. coli cells Site-specific mutagenesis Analysis on agarose gel Transformation of plasmids in E. coli Preparation

More information

4/3/2013. DNA Synthesis Replication of Bacterial DNA Replication of Bacterial DNA

4/3/2013. DNA Synthesis Replication of Bacterial DNA Replication of Bacterial DNA 4/3/03 3 4 5 6 7 8 9 0 Chapter 8 Microbial Genetics Terminology Genetics: The study of what genes are, how they carry information, how information is expressed, and how genes are replicated Gene: A segment

More information

Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward

Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward Gene Jockeying: Tricks of the Trade so that your Ligations work Every Time! Bevin Engelward We will first review a simple example of a ligation. Know Your Vectors! -Find out as much as you can about your

More information

Enzyme that uses RNA as a template to synthesize a complementary DNA

Enzyme that uses RNA as a template to synthesize a complementary DNA Biology 105: Introduction to Genetics PRACTICE FINAL EXAM 2006 Part I: Definitions Homology: Comparison of two or more protein or DNA sequence to ascertain similarities in sequences. If two genes have

More information

G0463 pscaavmcsbghpa MCS. Plasmid Features:

G0463 pscaavmcsbghpa MCS. Plasmid Features: 3200 G0463 pscaavmcsbghpa Plasmid Features: Coordinates Feature 980-1085 AAV2 ITR 106bp (mutated ITR) 1110-1226 MCS 1227-1440 BgHpA 1453-1595 AAV2 ITR (143bp) 2496-3356 B-lactamase (Ampicillin) Antibiotic

More information

HE Swift Cloning Kit

HE Swift Cloning Kit HE Swift Cloning Kit For high-efficient cloning of PCR products either blunt or sticky-end Kit Contents Contents VTT-BB05 phe Vector (35 ng/µl) 20 µl T4 DNA Ligase (3 U/µl) 20 µl 2 Reaction Buffer 100

More information

Biotechnology. Chapter 20. Biology Eighth Edition Neil Campbell and Jane Reece. PowerPoint Lecture Presentations for

Biotechnology. Chapter 20. Biology Eighth Edition Neil Campbell and Jane Reece. PowerPoint Lecture Presentations for Chapter 20 Biotechnology PowerPoint Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright

More information

Hetero-Stagger PCR Cloning Kit

Hetero-Stagger PCR Cloning Kit Product Name: Code No: Size: DynaExpress Hetero-Stagger PCR Cloning Kit DS150 20 reactions Kit Components: Box 1 (-20 ) phst-1 Vector, linearized Annealing Buffer Ligase Mixture phst Forward Sequence Primer

More information

Experimental genetics - I

Experimental genetics - I Experimental genetics - I Examples of diseases with genetic-links Hemophilia (complete loss or altered form of factor VIII): bleeding disorder Duchenne muscular dystrophy (altered form of dystrophin) muscle

More information

Chapter 6 - Molecular Genetic Techniques

Chapter 6 - Molecular Genetic Techniques Chapter 6 - Molecular Genetic Techniques Two objects of molecular & genetic technologies For analysis For generation Molecular genetic technologies! For analysis DNA gel electrophoresis Southern blotting

More information

Factors affecting PCR

Factors affecting PCR Lec. 11 Dr. Ahmed K. Ali Factors affecting PCR The sequences of the primers are critical to the success of the experiment, as are the precise temperatures used in the heating and cooling stages of the

More information

QUICK-Clone TM User Manual. cdna

QUICK-Clone TM User Manual. cdna QUICK-Clone TM User Manual cdna PT1150-1 (PR752268) Published 25 May 2007 Table of Contents I. Introduction 3 II. Applications Discussion 4 A. Primer Design 4 B. Setting up the PCR Reaction 4 C. Example

More information

Stratagene Mutagenesis Solutions for Your Protein Engineering Needs

Stratagene Mutagenesis Solutions for Your Protein Engineering Needs Stratagene Mutagenesis Solutions for Your Protein Engineering Needs Protein engineering via mutagenesis allows researchers to modulate protein activity and characterize structurefunction relationships,

More information

California Institute of Technology. Directed evolution. Dr. F.H. Arnold s lab

California Institute of Technology. Directed evolution. Dr. F.H. Arnold s lab Directed evolution. Dr. F.H. Arnold s lab May 4, 1999 Mutagenic PCR -[Mn] The amount of Mn used in the reaction should be titrated to produce the desired mutagenic rate. Libraries that have close to 30%

More information

MOLECULAR GENETICS: TRANSFORMATION AND CLONING adapted by Dr. D. L. Vogelien

MOLECULAR GENETICS: TRANSFORMATION AND CLONING adapted by Dr. D. L. Vogelien Introduction MOLECULAR GENETICS: TRANSFORMATION AND CLONING adapted by Dr. D. L. Vogelien The field of molecular genetics has resulted in a number of practical applications that have been of tremendous

More information

CHAPTER 9 DNA Technologies

CHAPTER 9 DNA Technologies CHAPTER 9 DNA Technologies Recombinant DNA Artificially created DNA that combines sequences that do not occur together in the nature Basis of much of the modern molecular biology Molecular cloning of genes

More information

Directe d Mutagenesis

Directe d Mutagenesis Directe d Mutagenesis A Practical Approac h M. J. McPHERSON 1. Mutagenesis facilitated by the removal or introduction of unique restriction sites 1 P. Carte r 1. Introduction to site-directed mutagenesis

More information

Molecular Biology: Gene cloning

Molecular Biology: Gene cloning Molecular Biology: Gene cloning Author: Prof Marinda Oosthuizen Licensed under a Creative Commons Attribution license. CLONING VECTORS The central component of a gene cloning experiment is the vector or

More information

CHAPTER 2A HOW DO YOU BEGIN TO CLONE A GENE? CHAPTER 2A STUDENT GUIDE 2013 Amgen Foundation. All rights reserved.

CHAPTER 2A HOW DO YOU BEGIN TO CLONE A GENE? CHAPTER 2A STUDENT GUIDE 2013 Amgen Foundation. All rights reserved. CHAPTER 2A HOW DO YOU BEGIN TO CLONE A GENE? 35 INTRODUCTION In the Program Introduction, you learned that the increase in diabetes in the United States has resulted in a great demand for its treatment,

More information

Problem Set 8. Answer Key

Problem Set 8. Answer Key MCB 102 University of California, Berkeley August 11, 2009 Isabelle Philipp Online Document Problem Set 8 Answer Key 1. The Genetic Code (a) Are all amino acids encoded by the same number of codons? no

More information

Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:20-25 Copyright December 2004, M&I UBC

Journal of Experimental Microbiology and Immunology (JEMI) Vol. 6:20-25 Copyright December 2004, M&I UBC Preparing Plasmid Constructs to Investigate the Characteristics of Thiol Reductase and Flavin Reductase With Regard to Solubilizing Insoluble Proteinase Inhibitor 2 in Bacterial Protein Overexpression

More information

Polymerase Chain Reaction PCR

Polymerase Chain Reaction PCR Polymerase Chain Reaction PCR What is PCR? An in vitro process that detects, identifies, and copies (amplifies) a specific piece of DNA in a biological sample. Discovered by Dr. Kary Mullis in 1983. A

More information

2054, Chap. 14, page 1

2054, Chap. 14, page 1 2054, Chap. 14, page 1 I. Recombinant DNA technology (Chapter 14) A. recombinant DNA technology = collection of methods used to perform genetic engineering 1. genetic engineering = deliberate modification

More information

Recombinant DNA Technology

Recombinant DNA Technology History of recombinant DNA technology Recombinant DNA Technology (DNA cloning) Majid Mojarrad Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists

More information

DNA REPLICATION & BIOTECHNOLOGY Biology Study Review

DNA REPLICATION & BIOTECHNOLOGY Biology Study Review DNA REPLICATION & BIOTECHNOLOGY Biology Study Review DNA DNA is found in, in the nucleus. It controls cellular activity by regulating the production of, which includes It is a very long molecule made up

More information

I. Gene Cloning & Recombinant DNA. Biotechnology: Figure 1: Restriction Enzyme Activity. Restriction Enzyme:

I. Gene Cloning & Recombinant DNA. Biotechnology: Figure 1: Restriction Enzyme Activity. Restriction Enzyme: I. Gene Cloning & Recombinant DNA Biotechnology: Figure 1: Restriction Enzyme Activity Restriction Enzyme: Most restriction enzymes recognize a single short base sequence, or Restriction Site. Restriction

More information

Exam 2 Key - Spring 2008 A#: Please see us if you have any questions!

Exam 2 Key - Spring 2008 A#: Please see us if you have any questions! Page 1 of 5 Exam 2 Key - Spring 2008 A#: Please see us if you have any questions! 1. A mutation in which parts of two nonhomologous chromosomes change places is called a(n) A. translocation. B. transition.

More information

Enzymatic assembly of DNA molecules up to several hundred kilobases

Enzymatic assembly of DNA molecules up to several hundred kilobases nature methods Enzymatic assembly of DNA molecules up to several hundred kilobases Daniel G Gibson, Lei Young, Ray-Yuan Chuang, J Craig Venter, Clyde A Hutchison III & Hamilton O Smith Supplementary figures

More information

d. reading a DNA strand and making a complementary messenger RNA

d. reading a DNA strand and making a complementary messenger RNA Biol/ MBios 301 (General Genetics) Spring 2003 Second Midterm Examination A (100 points possible) Key April 1, 2003 10 Multiple Choice Questions-4 pts. each (Choose the best answer) 1. Transcription involves:

More information

Fast and efficient site-directed mutagenesis with Platinum SuperFi DNA Polymerase

Fast and efficient site-directed mutagenesis with Platinum SuperFi DNA Polymerase APPLICATION NOTE Platinum Superi Polymerase ast and efficient site-directed mutagenesis with Platinum Superi Polymerase Introduction Site-directed mutagenesis is one of the most essential techniques to

More information

GenBuilder TM Cloning Kit User Manual

GenBuilder TM Cloning Kit User Manual GenBuilder TM Cloning Kit User Manual Cat.no L00701 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2 Ⅱ. DNA

More information

Welcome to Class 18! Lecture 18: Outline and Objectives. Replication is semiconservative! Replication: DNA DNA! Introductory Biochemistry!

Welcome to Class 18! Lecture 18: Outline and Objectives. Replication is semiconservative! Replication: DNA DNA! Introductory Biochemistry! Lecture 18: Outline and Objectives Welcome to Class 18! Introductory Biochemistry! l DNA Replication! l DNA polymerase! l the enzymatic reaction! l proofreading and accuracy! l DNA synthesis! l origins

More information

NZYGene Synthesis kit

NZYGene Synthesis kit Kit components Component Concentration Amount NZYGene Synthesis kit Catalogue number: MB33901, 10 reactions GS DNA Polymerase 1U/ μl 30 μl Reaction Buffer for GS DNA Polymerase 10 150 μl dntp mix 2 mm

More information

BEST QUALITY HIGHEST PURITY. Recombinant ENZYMES & PROTEINS

BEST QUALITY HIGHEST PURITY. Recombinant ENZYMES & PROTEINS BEST QUALITY HIGHEST PURITY Recombinant ENZYMES & PROTEINS We offer a wide range of highest quality enzymes and proteins for molecular biology including DNA polymerases, reverse transcriptases, DNA ligases,

More information