Recombinant DNA Technology. Recombinant DNA Technology. Learning Objective. Molecular & Cell Biology
|
|
- Juniper Warren
- 7 years ago
- Views:
Transcription
1 Recombinant DNA technology which came into existence in the 1970s, allows for genetic manipulation of organisms by incorporating DNA sequences from different sources into a single recombinant molecule. This revolutionary technology has opened up several applications in plant genomics and clinical research. Learning Objective After interacting with this Learning Object, the learner will be able to, ü List out tools used for gene exploration. ü Utilize the knowledge on creation of a genomic library. ü Recall about transgenic plants & animals.
2 Tools used for gene exploration Restriction endonucleases cleave double stranded DNA at specific recognition sequences which can be used for isolating genes and cloning new DNA molecules. These enzymes are typically found in prokaryotes where they function as a defence mechanism against foreign DNA. Recognitions sites are typically 4-8 base pairs long and are very often palindromic i.e. they read the same in both directions. Cleavage at a phosphodiester bond can produce either blunt ends or cohesive ends. Two DNA molecules that have been cleaved by the same restriction enzyme can be ligated using DNA ligase to produce a recombinant molecule. The fragments may also be separated using electrophoresis techniques.
3 Tools used for gene exploration DNA that has been fragmented by restriction enzymes can be separated by agarose gel electrophoresis. The DNA is then denatured and blotted onto a nitrocellulose sheet such that their positions remain intact, a process known as Southern blotting. The DNA strands are then probed with a radiolableled DNA molecule which hybridizes to the complementary sequence and can be detected by autoradiography. A similar procedure for RNA molecules is referred to as Northern blotting.
4 Tools used for gene exploration The polymerase chain reaction can be used to amplify specific DNA sequences of interest. The reaction consists of multiple cycles of strand separation, primer annealing and strand elongation to generate millions of copies of the target sequence within very short time. The two parent DNA strands are first separated by heating them to 95oC after which large excess of primers are added and the solution cooled abruptly (e.g. 54oC), thereby allowing the primers to anneal to each end of the target strand. Strand elongation then takes place at 72oC with help of heat-stable DNA Polymerase from Thermus aquaticus. Both primers are elongated in 5 to 3 direction beyond the target sequence.
5 Tools used for gene exploration Multiple rounds of PCR are carried out in this manner with repeated cycles of strand separation, primer hybridization and strand elongation. It is possible to carry out these sequence of reactions continuously in a closed container without further addition of reagents after the first cycle, simply by modifying the temperature of the reaction mixture. Although the first cycle yields strands that are longer than the target sequence, only the target is amplified from the end of the second cycle onwards. The sequence gets amplified 2n-fold at the end of n cycles. This is an extremely useful technique in diagnostics, forensics and molecular evolution.
6 Tools used for gene exploration A simple and elegant method for DNA sequencing was devised by Frederick Sanger where a collection of DNA fragments are synthesized by means of controlled interruption of enzymatic replication. Four DNA synthesis reactions are carried out simultaneously with the strand whose sequence is to be determined being used as the template. The reaction mixture consists of regular deoxy nucleotides and DNA Polymerase along with a small amount of one labelled dideoxy nucleotide analog being added to each of the four reaction mixtures. A primer is added to begin the DNA synthesis and strand elongation continues until a dideoxy analog gets added instead of the regular dntp. Chain termination occurs at this stage due to the absence of a 3 OH group for formation of the next phosphodiester bond.
7 Tools used for gene exploration The synthesized strands are separated from each other, after which the differentially labelled strands of various lengths are separated by electrophoresis. The smallest fragments move further in the gel while the larger fragments remain close to the point of application. The different fluorescent labels of each ddntp can then be detected by scanning the gel with a beam of laser. The output sequence obtained is complementary to the template strand, which can be used to deduce the original desired template sequence.
8 Creation of a genomic library Genomic DNA and DNA of the vector molecule are cleaved with the same restriction enzyme so as to generate complimentary end sequences. These are then ligated by means of the enzyme DNA ligase to generate recombinant DNA molecules.
9 Creation of a genomic library These recombinant molecules can be packaged in-vitro into suitable phage particles which serve as useful vectors to carry the foreign DNA molecules. Lambda and M13 are two of the most commonly used phage particles for this purpose. DNA inserts upto 10 kilobases can be inserted into these phage particles.
10 Creation of a genomic library There are other vector molecules that can take up DNA fragments from genomic DNA. Plasmids possess sites for various restriction enzymes as well as antibiotic resistance sites which help for screening purposes. Certain plasmids like pbr322 can only take up smaller DNA inserts upto ~ 10kB while larger plasmids known as bacterial artificial chromosomes can take up larger inserts upto 300 kb. The Yeast artificial chromosome is a eukaryotic vector that can take up large DNA inserts and consists of restriction sites, centromer and two telomeric sites.
11 Creation of a genomic library The phage molecules carrying the recombinant DNA with genomic inserts are used to infect E. coli cells. These molecules then get amplified with each round of replication of the E. coli cell. This collection of bacterial cells harboring the various genomic DNA fragments is known as the genomic library.
12 Creation of a genomic library Once the library has been created, it is essential to have techniques to retrieve a sequence of interest. The transformed cells are selected and plated on agar, after which the plate is blotted on to a nitrocellulose paper. This is then treated with alkali, bringing about disruption of the cells and denaturation of the dsdna. The denatured DNA is then probed for the sequence of interest by a radiolabelled molecule having a complementary sequence.
13 Transgenic plants & animals When a plant gets injured, there is release of the phenolic compound acetosyringone, which is detected by Agrobacterium tumefaciens. Upon detection, the virulence genes of tumor-inducing (Ti) plasmid get expressed which encode the enzymes that are essential for transfer of the T DNA into the nucleus of the plant cell. Once the T-DNA gets integrated with the plant chromosome there is release of cytokinins, auxins etc. which brings about tumor formation in the plant.
14 Transgenic plants & animals The useful property of infection by Agrobacterium has allowed several foreign genes of interest to be introduced into plant cells as per the requirement. One plasmid of the cell is the Ti plasmid without the T-DNA. The other plasmid contains the gene of interest along with antibiotic resistance genes placed in between two repeat units that are essential for gene transfer. The gene of interest such as genes for pesticide resistance, better yield etc. invades the plant at the site of injury. Once this happens, the foreign gene gets inserted into the plant DNA, which is confirmed by plating on to agar containing the suitable antibiotic. Only those which have taken up the gene will grow on such plates.
15 Transgenic plants & animals Transgenic animals especially mice are commonly used to express foreign DNA molecules. This is often done with the help of retroviruses. Successful expression of these inserts in larger mammals has allowed the development of several recombinant products useful for medicine.
16 Tools used for gene exploration 1. Restriction analysis: A molecular biology technique that is used to fragment and manipulate DNA into manageable lengths before further analysis. The following components are required for restriction analysis. a) Restriction enzymes: An enzyme that cleaves single or double stranded DNA at specific locations based on the nucleotide sequences recognized. b) Restriction sites: These are regions of the nucleotide sequence that are recognised by the restriction enzymes and subsequently cleaved. c) DNA Ligase: Enzyme that is involved in repairing or joining single stranded breaks or discontinuities in double stranded DNA. d) Cohesive ends: A cohesive or sticky end of DNA refers to those DNA molecules having a 3 or 5 overhang region after they have been cleaved by the restriction enzyme. These overhangs possess nucleotide sequences that have complementary regions and can therefore easily anneal together. e) Blunt ends: Blunt ends of DNA molecules refer to those that do not possess any overhang region but are instead cleaved at the same nucleotide position on both strands thereby producing flat or blunt ends.
17 Tools used for gene exploration 2. Blotting techniques: These are techniques that have been developed to characterize DNA and RNA. The Southern blot, developed by Edwin Southern, is used to characterize DNA while the Northern blot is used for RNA. a) Electrophoresis: Electrophoresis is a gelbased analytical technique that is used for separation and visualization of biomolecules like DNA, RNA and proteins based on their fragment lengths or charge-to-mass ratios using an electric field. b) Agarose gel: Gels made of agarose are commonly used for separation of nucleotide molecules. These are made up of 0.7-2% agarose dissolved in the electrophoresis buffer but concentrations can vary depending on the length of fragments to be separated. c) Blotting: The process by which nucleotide molecules separated on the electrophoresis gel are transferred on to another surface such as nitrocellulose by placing them in contact with each other. d) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which single stranded DNA molecules separated by electrophoresis are transferred for further probing and analysis. e) Probing & autoradiography: The single stranded DNA molecules bound to the nitrocellulose membrane are probed for specific sequences by means of a radioactive probe molecule. Excess probe molecules are washed off after which the radioactivity is detected by means of autoradiography. f) Autoradiograph: The image pattern produced due to decay emissions from a radioactive material.
18 Tools used for gene exploration 3. Polymerase Chain reaction (PCR): The polymerase chain reaction is an extremely useful molecular biology tool that allows for amplification of a segment of DNA upto billionfold. This helps in characterization and manipulation of very small quantities of DNA and has found several diverse applications. a) Target sequence: The sequence of interest of DNA that is to be amplified. b) PCR cycle: One round or cycle of PCR refers to strand separation at 95oC, annealing of primers at 54oC and elongation of these primers at 72oC by the polymerase enzyme. Every PCR cycle results in doubling of the number of strands present. c) Primers: A short strand of nucleotides that acts as a starting point for DNA synthesis by providing a free 3 -OH end. These are hybridized to the target DNA and then elongated by means of the polymerase. d) Taq polymerase: This is a thermostable Polymerase enzyme that is obtained from bacterium Thermus aquaticus. It is used for reactions due to its ability to perform elongation even at high temperatures. DNA the PCR DNA
19 Creation of a genomic library 1. Genomic library: A collection of host bacteria, each carrying a DNA fragment as part of a cloning vector, such that the entire set of DNA fragments when assembled represents the complete genome of an organism. 2. Genomic DNA: The entire DNA sequence of all chromosomes of an organism constitutes its genomic DNA. Genome sequencing projects aimed at deciphering the complete genome sequence of organisms including humans. 3. Splicing & ligation: The process by which the genomic DNA of interest is broken down into fragments using restriction enzymes and then inserted into a suitable vector where it is joined together or ligated using a DNA ligase enzyme. 4. Vector DNA: A vector is a DNA molecule that acts as a vehicle or medium to transfer foreign DNA into another cell. Commonly used vectors include plasmids, phages, bacterial or yeast artificial chromosomes. The DNA fragment of interest is inserted into this vector molecule by means of suitable enzymes. 5. In-vitro packaging: Process by which the vector DNA containing the genomic fragments are taken up by the phage particles and packaged such that they have a protective coating around them. 6. Phage particles: These are viruses that are capable of taking up foreign DNA and infecting bacterial cells, thereby transferring the DNA into them.
20 Creation of a genomic library 7. Infection: The process by which the phage particles attach themselves to the bacterial cells and insert their genetic material into them. 8. E. coli cells: One of the most commonly used bacterial cells for creation of genomic libraries due to their simple growth requirements and ease of manipulation.
21 Transgenic plants & animals 1. Ti plasmid: Tumour-inducing or Ti plasmids are plasmid molecules that are carried by the soil bacterium Agrobacterium tumefaciens. This plasmid contains genes that can bring about development of tumour state and therefore synthesis of opines. 5. Crown gall: The lump of tumour tissue that is formed at the site of plant infection upon insertion of genes from the Ti plasmid by the Agrobacterium is known as the Crown gall. 2. T-DNA: The small 20 kb DNA insert of Ti plasmid that gets integrated into the genome of infected plant cells is known as the T-DNA. 6. Release of cytokines, auxins & opines: Formation of the tumour results in release of compounds such as cytokines, auxins and opines, which are metabolized by the infecting bacteria thereby disrupting the plant cell metabolism. 3. Agrobacterium cell: This is a common soil bacterium known as Agrobacterium tumefaciens that is capable of introducing foreign genes into plant cells by means of its plasmid. 7. Transgenic mice: Many transgenic animals have been developed that harbour and express foreign genes of interest. The most commonly used animals are mice due to ease of handling and their convenient growth cycle. 4. vir genes: These are the virulence genes carried by the plasmid that are essential for infection of the plant cells.
22
23
24
25
26
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 informationrestriction enzymes 350 Home R. Ward: Spring 2001
restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually
More informationRecombinant 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 informationBiotechnology: DNA Technology & Genomics
Chapter 20. Biotechnology: DNA Technology & Genomics 2003-2004 The BIG Questions How can we use our knowledge of DNA to: diagnose disease or defect? cure disease or defect? change/improve organisms? What
More informationRecombinant DNA and Biotechnology
Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study
More informationGenetic Engineering and Biotechnology
1 So, what is biotechnology?? The use of living organisms to carry out defined chemical processes for industrial or commercial application. The office of Technology Assessment of the U.S. Congress defines
More informationCHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA
CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the
More informationBiotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College
Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology
More informationHCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise:
HCS604.03 Exercise 1 Dr. Jones Spring 2005 Recombinant DNA (Molecular Cloning) exercise: The purpose of this exercise is to learn techniques used to create recombinant DNA or clone genes. You will clone
More informationDNA Fingerprinting. Unless they are identical twins, individuals have unique DNA
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
More informationGene Cloning. Reference. T.A. Brown, Gene Cloning, Chapman and Hall. S.B. Primrose, Molecular Biotechnology, Blackwell
Gene Cloning 2004 Seungwook Kim Chem. & Bio. Eng. Reference T.A. Brown, Gene Cloning, Chapman and Hall S.B. Primrose, Molecular Biotechnology, Blackwell Why Gene Cloning is Important? A century ago, Gregor
More informationHiPer 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 informationLecture 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 informationCCR Biology - Chapter 9 Practice Test - Summer 2012
Name: Class: Date: CCR Biology - Chapter 9 Practice Test - Summer 2012 Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Genetic engineering is possible
More informationDNA Scissors: Introduction to Restriction Enzymes
DNA Scissors: Introduction to Restriction Enzymes Objectives At the end of this activity, students should be able to 1. Describe a typical restriction site as a 4- or 6-base- pair palindrome; 2. Describe
More informationGene Mapping Techniques
Gene Mapping Techniques OBJECTIVES By the end of this session the student should be able to: Define genetic linkage and recombinant frequency State how genetic distance may be estimated State how restriction
More informationThe 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 information1/12 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 information2. The number of different kinds of nucleotides present in any DNA molecule is A) four B) six C) two D) three
Chem 121 Chapter 22. Nucleic Acids 1. Any given nucleotide in a nucleic acid contains A) two bases and a sugar. B) one sugar, two bases and one phosphate. C) two sugars and one phosphate. D) one sugar,
More informationBasic Concepts Recombinant DNA Use with Chapter 13, Section 13.2
Name Date lass Master 19 Basic oncepts Recombinant DN Use with hapter, Section.2 Formation of Recombinant DN ut leavage Splicing opyright lencoe/mcraw-hill, a division of he Mcraw-Hill ompanies, Inc. Bacterial
More informationAppendix 2 Molecular Biology Core Curriculum. Websites and Other Resources
Appendix 2 Molecular Biology Core Curriculum Websites and Other Resources Chapter 1 - The Molecular Basis of Cancer 1. Inside Cancer http://www.insidecancer.org/ From the Dolan DNA Learning Center Cold
More information4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true?
Chapter 25 DNA Metabolism Multiple Choice Questions 1. DNA replication Page: 977 Difficulty: 2 Ans: C The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis.
More informationHow 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 informationTransfection-Transfer of non-viral genetic material into eukaryotic cells. Infection/ Transduction- Transfer of viral genetic material into cells.
Transfection Key words: Transient transfection, Stable transfection, transfection methods, vector, plasmid, origin of replication, reporter gene/ protein, cloning site, promoter and enhancer, signal peptide,
More informationForensic DNA Testing Terminology
Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.
More informationRecombinant DNA Unit Exam
Recombinant DNA Unit Exam Question 1 Restriction enzymes are extensively used in molecular biology. Below are the recognition sites of two of these enzymes, BamHI and BclI. a) BamHI, cleaves after the
More informationCompiled and/or written by Amy B. Vento and David R. Gillum
Fact Sheet Describing Recombinant DNA and Elements Utilizing Recombinant DNA Such as Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology Compiled and/or written
More informationBacterial Transformation and Plasmid Purification. Chapter 5: Background
Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment
More informationNucleic Acid Techniques in Bacterial Systematics
Nucleic Acid Techniques in Bacterial Systematics Edited by Erko Stackebrandt Department of Microbiology University of Queensland St Lucia, Australia and Michael Goodfellow Department of Microbiology University
More informationExpression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu
Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.
More informationGENE CLONING AND RECOMBINANT DNA TECHNOLOGY
GENE CLONING AND RECOMBINANT DNA TECHNOLOGY What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning.
More information2. 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 informationStructure and Function of DNA
Structure and Function of DNA DNA and RNA Structure DNA and RNA are nucleic acids. They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. The four
More information- In 1976 1977, Allan Maxam and walter Gilbert devised the first method for sequencing DNA fragments containing up to ~ 500 nucleotides.
DNA Sequencing - DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA. -
More informationGenetic Technology. Name: Class: Date: Multiple Choice Identify the choice that best completes the statement or answers the question.
Name: Class: Date: Genetic Technology Multiple Choice Identify the choice that best completes the statement or answers the question. 1. An application of using DNA technology to help environmental scientists
More information1. Molecular computation uses molecules to represent information and molecular processes to implement information processing.
Chapter IV Molecular Computation These lecture notes are exclusively for the use of students in Prof. MacLennan s Unconventional Computation course. c 2013, B. J. MacLennan, EECS, University of Tennessee,
More informationAn Overview of DNA Sequencing
An Overview of DNA Sequencing Prokaryotic DNA Plasmid http://en.wikipedia.org/wiki/image:prokaryote_cell_diagram.svg Eukaryotic DNA http://en.wikipedia.org/wiki/image:plant_cell_structure_svg.svg DNA Structure
More informationCloning GFP into Mammalian cells
Protocol for Cloning GFP into Mammalian cells Studiepraktik 2013 Molecular Biology and Molecular Medicine Aarhus University Produced by the instructors: Tobias Holm Bønnelykke, Rikke Mouridsen, Steffan
More informationDNA Replication in Prokaryotes
OpenStax-CNX module: m44488 1 DNA Replication in Prokaryotes OpenStax College This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 3.0 By the end of this section,
More informationIMBB 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 informationCLONING IN ESCHERICHIA COLI
CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a
More informationThe Biotechnology Education Company
EDVTEK P.. Box 1232 West Bethesda, MD 20827-1232 The Biotechnology 106 EDV-Kit # Principles of DNA Sequencing Experiment bjective: The objective of this experiment is to develop an understanding of DNA
More informationAP BIOLOGY 2007 SCORING GUIDELINES
AP BIOLOGY 2007 SCORING GUIDELINES Question 4 A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with two restriction enzymes in three separate treatments: EcoRI, HaeIII,
More informationEvery time a cell divides the genome must be duplicated and passed on to the offspring. That is:
DNA Every time a cell divides the genome must be duplicated and passed on to the offspring. That is: Original molecule yields 2 molecules following DNA replication. Our topic in this section is how is
More informationReal-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 informationDNA Technology Mapping a plasmid digesting How do restriction enzymes work?
DNA Technology Mapping a plasmid A first step in working with DNA is mapping the DNA molecule. One way to do this is to use restriction enzymes (restriction endonucleases) that are naturally found in bacteria
More informationVLLM0421c Medical Microbiology I, practical sessions. Protocol to topic J10
Topic J10+11: Molecular-biological methods + Clinical virology I (hepatitis A, B & C, HIV) To study: PCR, ELISA, your own notes from serology reactions Task J10/1: DNA isolation of the etiological agent
More informationSTUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS
STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY
More informationPrimeSTAR 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 informationSection 16.1 Producing DNA fragments
Section 16.1 Producing DNA fragments Recombinant DNA combined DNA of two different organisms The process of using DNA technology to make certain proteins is as follows: 1.) Isolation of the DNA fragments
More informationDNA Sequence Analysis
DNA Sequence Analysis Two general kinds of analysis Screen for one of a set of known sequences Determine the sequence even if it is novel Screening for a known sequence usually involves an oligonucleotide
More information2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line
i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) 1 1.1.1 Pathogenesis of Hepatitis B 1 1.1.2 Genome organization of HBV 3 1.1.3 Structure of HBV virion 5 1.1.4 HBV life cycle 5 1.1.5 Experimental models
More informationFirst 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 informationPyroPhage 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 informationBio 102 Practice Problems Recombinant DNA and Biotechnology
Bio 102 Practice Problems Recombinant DNA and Biotechnology Multiple choice: Unless otherwise directed, circle the one best answer: 1. Which of the following DNA sequences could be the recognition site
More informationINTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B
INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS
More informationTaq98 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 informationNext Generation Sequencing
Next Generation Sequencing DNA sequence represents a single format onto which a broad range of biological phenomena can be projected for high-throughput data collection Over the past three years, massively
More informationTroubleshooting 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 informationHOUR EXAM 1: September 29, 2009 (Tuesday) EXAM WILL COVER: EXAM 1 REVIEW: Monday, Sept. 28, 2008, 5-6:00 PM, BSW208
HOUR EXAM 1: September 29, 2009 (Tuesday) EXAM WILL COVER: CHAPTER 25 CHAPER 4 CHAPTER 5 TO END of Sept 24 Lecture EXAM 1 REVIEW: Monday, Sept. 28, 2008, 5-6:00 PM, BSW208 Sept 18, 2008 CHAPTER 5 Exploring
More informationTranscription in prokaryotes. Elongation and termination
Transcription in prokaryotes Elongation and termination After initiation the σ factor leaves the scene. Core polymerase is conducting the elongation of the chain. The core polymerase contains main nucleotide
More informationHow is genome sequencing done?
How is genome sequencing done? Using 454 Sequencing on the Genome Sequencer FLX System, DNA from a genome is converted into sequence data through four primary steps: Step One DNA sample preparation; Step
More informationChapter 6 DNA Replication
Chapter 6 DNA Replication Each strand of the DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Each strand can therefore
More informationIIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR)
IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR) Background Infectious diseases caused by pathogenic organisms such as bacteria, viruses, protozoa,
More informationGenetics Test Biology I
Genetics Test Biology I Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Avery s experiments showed that bacteria are transformed by a. RNA. c. proteins.
More informationRT31-020 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 informationThe E. coli Insulin Factory
The E. coli Insulin Factory BACKGROUND Bacteria have not only their normal DNA, they also have pieces of circular DNA called plasmids. Plasmids are a wonderfully ally for biologists who desire to get bacteria
More informationCloning 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 informationPharmaceutical Biotechnology. Recombinant DNA technology Western blotting and SDS-PAGE
Pharmaceutical Biotechnology Recombinant DNA technology Western blotting and SDS-PAGE Recombinant DNA Technology Protein Synthesis Western Blot Western blots allow investigators to determine the molecular
More informationMolecular Cloning, Product Brochure
, Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0)26 326 44 50 T BE +32 (0)2 503 03 48 Begonialaan 3a F NL +31 (0)26 326 44 51 F BE
More informationGenetics 301 Sample Final Examination Spring 2003
Genetics 301 Sample Final Examination Spring 2003 50 Multiple Choice Questions-(Choose the best answer) 1. A cross between two true breeding lines one with dark blue flowers and one with bright white flowers
More informationGenetics Module B, Anchor 3
Genetics Module B, Anchor 3 Key Concepts: - An individual s characteristics are determines by factors that are passed from one parental generation to the next. - During gamete formation, the alleles for
More informationReplication Study Guide
Replication Study Guide This study guide is a written version of the material you have seen presented in the replication unit. Self-reproduction is a function of life that human-engineered systems have
More informationMolecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION
Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN
More informationGenomic DNA Clean & Concentrator Catalog Nos. D4010 & D4011
Page 0 INSTRUCTION MANUAL Catalog Nos. D4010 & D4011 Highlights Quick (5 minute) spin column recovery of large-sized DNA (e.g., genomic, mitochondrial, plasmid (BAC/PAC), viral, phage, (wga)dna, etc.)
More informationDNA sequencing. Dideoxy-terminating sequencing or Sanger dideoxy sequencing
DNA sequencing Dideoxy-terminating sequencing or Sanger dideoxy sequencing Tools DNA template (single stranded) Specific primer (usually 17-23 mer, free 3 -OH) dntps DNA polymerase capacity of polymerizing
More informationCentral Dogma. Lecture 10. Discussing DNA replication. DNA Replication. DNA mutation and repair. Transcription
Central Dogma transcription translation DNA RNA Protein replication Discussing DNA replication (Nucleus of eukaryote, cytoplasm of prokaryote) Recall Replication is semi-conservative and bidirectional
More informationDNA PROFILING IN FORENSIC SCIENCE
DA PROFILIG I FORESIC SCIECE DA is the chemical code that is found in every cell of an individual's body, and is unique to each individual. Because it is unique, the ability to examine DA found at a crime
More informationName Class Date. Figure 13 1. 2. Which nucleotide in Figure 13 1 indicates the nucleic acid above is RNA? a. uracil c. cytosine b. guanine d.
13 Multiple Choice RNA and Protein Synthesis Chapter Test A Write the letter that best answers the question or completes the statement on the line provided. 1. Which of the following are found in both
More informationEssentials 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 informationSample Questions for Exam 3
Sample Questions for Exam 3 1. All of the following occur during prometaphase of mitosis in animal cells except a. the centrioles move toward opposite poles. b. the nucleolus can no longer be seen. c.
More informationWhy Gene Cloning and DNA Analysis are Important
Chapter 1 Why Gene Cloning and DNA Analysis are Important 3 What is per'i, 6 Why gene cloning and per are so chain reaction, 4 important, 8 What is gene.5 How to find your way through this book, 12 In
More informationTransformAid Bacterial Transformation Kit
Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection
More informationDNA (genetic information in genes) RNA (copies of genes) proteins (functional molecules) directionality along the backbone 5 (phosphate) to 3 (OH)
DNA, RNA, replication, translation, and transcription Overview Recall the central dogma of biology: DNA (genetic information in genes) RNA (copies of genes) proteins (functional molecules) DNA structure
More informationDescription: Molecular Biology Services and DNA Sequencing
Description: Molecular Biology s and DNA Sequencing DNA Sequencing s Single Pass Sequencing Sequence data only, for plasmids or PCR products Plasmid DNA or PCR products Plasmid DNA: 20 100 ng/μl PCR Product:
More informationAmazing DNA facts. Hands-on DNA: A Question of Taste Amazing facts and quiz questions
Amazing DNA facts These facts can form the basis of a quiz (for example, how many base pairs are there in the human genome?). Students should be familiar with most of this material, so the quiz could be
More informationChapter 3 Contd. Western blotting & SDS PAGE
Chapter 3 Contd. Western blotting & SDS PAGE Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different
More informationTechniques in Molecular Biology (to study the function of genes)
Techniques in Molecular Biology (to study the function of genes) Analysis of nucleic acids: Polymerase chain reaction (PCR) Gel electrophoresis Blotting techniques (Northern, Southern) Gene expression
More informationEuropean Medicines Agency
European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein
More informationMilestones of bacterial genetic research:
Milestones of bacterial genetic research: 1944 Avery's pneumococcal transformation experiment shows that DNA is the hereditary material 1946 Lederberg & Tatum describes bacterial conjugation using biochemical
More informationObjectives: Vocabulary:
Introduction to Agarose Gel Electrophoresis: A Precursor to Cornell Institute for Biology Teacher s lab Author: Jennifer Weiser and Laura Austen Date Created: 2010 Subject: Molecular Biology and Genetics
More informationLAB 7 DNA RESTRICTION for CLONING
BIOTECHNOLOGY I DNA RESTRICTION FOR CLONING LAB 7 DNA RESTRICTION for CLONING STUDENT GUIDE GOALS The goals of this lab are to provide the biotech student with experience in DNA digestion with restriction
More informationGenetics Lecture Notes 7.03 2005. Lectures 1 2
Genetics Lecture Notes 7.03 2005 Lectures 1 2 Lecture 1 We will begin this course with the question: What is a gene? This question will take us four lectures to answer because there are actually several
More information1.5 page 3 DNA Replication S. Preston 1
AS Unit 1: Basic Biochemistry and Cell Organisation Name: Date: Topic 1.5 Nucleic Acids and their functions Page 3 l. DNA Replication 1. Go through PowerPoint 2. Read notes p2 and then watch the animation
More informationPicoMaxx 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 informationDNA CLONING. DNA segment has been developed: polymerase chain reaction PCR. Viral DNA-s bacteriophage λ, filamentous bacteriophages
DNA CLONING - What is cloning? The isolation of discrete pieces of DNA from their host organism and their amplification through propagation in the same or a different host More recently an alternitive,
More informationDNA Replication & Protein Synthesis. This isn t a baaaaaaaddd chapter!!!
DNA Replication & Protein Synthesis This isn t a baaaaaaaddd chapter!!! The Discovery of DNA s Structure Watson and Crick s discovery of DNA s structure was based on almost fifty years of research by other
More informationSTRUCTURES OF NUCLEIC ACIDS
CHAPTER 2 STRUCTURES OF NUCLEIC ACIDS What is the chemical structure of a deoxyribonucleic acid (DNA) molecule? DNA is a polymer of deoxyribonucleotides. All nucleic acids consist of nucleotides as building
More informationGenetic Analysis. Phenotype analysis: biological-biochemical analysis. Genotype analysis: molecular and physical analysis
Genetic Analysis Phenotype analysis: biological-biochemical analysis Behaviour under specific environmental conditions Behaviour of specific genetic configurations Behaviour of progeny in crosses - Genotype
More informationIntroduction 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