Recombinant DNA Technology Overview
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Recombinant DNA Technology Overview

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Which classification of plasmids is responsible for the sexual transfer of genetic material between bacteria?

  • Conjugative plasmids (correct)
  • Col-plasmids
  • Resistance-(R) plasmids
  • Degradative plasmids

    • What is the primary function of fertility (F) plasmids?

  • To kill other bacteria
  • To confer antibiotic resistance
  • To digest unusual substances
  • To facilitate bacterial conjugation (correct)

    • Which of the following statements is true regarding the size of plasmids?

  • Plasmids can only be found in Bacteria
  • Plasmids are generally larger than 10 kb
  • Plasmids are typically between 1 to 4 kb (correct)
  • Plasmids are exclusively found in Eukarya

    • What mechanism allows plasmids to replicate independently of the bacterial chromosome?

    <p>Independent origin of replication</p> Signup and view all the answers

    Which type of plasmid contains genes that provide resistance against antibiotics?

    <p>Resistance-(R) plasmids</p> Signup and view all the answers

    What role do degrative plasmids play in bacterial function?

    <p>Facilitate digestion of unusual substances</p> Signup and view all the answers

    What does a high copy number of a plasmid indicate?

    <p>The plasmid is present in many copies per bacterial cell</p> Signup and view all the answers

    Which type of plasmid would likely be used in genetic engineering as a vector?

    <p>Resistance-(R) plasmids</p> Signup and view all the answers

    What is the primary purpose of directional cloning in recombinant DNA technology?

    <p>To enhance the efficiency of ligation</p> Signup and view all the answers

    Which method is used in replica plating to screen transformants?

    <p>Selection on antibiotic media</p> Signup and view all the answers

    What role do competent cells play in the process of transformation?

    <p>They are capable of taking up foreign DNA from their environment.</p> Signup and view all the answers

    What type of plasmids are commonly used for cloning and expression in the lab?

    <p>PUC plasmids</p> Signup and view all the answers

    Which of the following is a key feature in the screening of transformants?

    <p>Using plasmids with color indicators</p> Signup and view all the answers

    Which replication enzyme is primarily used by most coli plasmids?

    <p>DNA Polymerase III</p> Signup and view all the answers

    What role does Rep protein play in plasmid replication?

    <p>Initiates plasmid replication</p> Signup and view all the answers

    Which of the following is NOT a requirement of a vector for cloning?

    <p>Inhibitory sequences</p> Signup and view all the answers

    What is the primary mechanism by which recombinant DNA is introduced into a host cell?

    <p>Transformation</p> Signup and view all the answers

    Which step in the recombinant DNA process involves joining vector and target DNA?

    <p>Cloning</p> Signup and view all the answers

    What is a key advantage of using restriction enzymes in the creation of recombinant DNA?

    <p>They cut DNA at specific sequences</p> Signup and view all the answers

    Which of the following refers to the process of isolating the gene and vector for cloning?

    <p>Cutting</p> Signup and view all the answers

    What is a plasmid's primary function in gene cloning?

    <p>To serve as a self-replicating vector</p> Signup and view all the answers

    What is the role of a selectable marker in a cloning vector?

    <p>To allow for screening of successful transformants</p> Signup and view all the answers

    What is the key reason for using vectors in recombinant DNA technology?

    <p>To ensure the DNA is passed onto daughter cells</p> Signup and view all the answers

    What is the main purpose of using ampicillin in the screening process of transformants?

    <p>To kill off any cells that do not contain the plasmid.</p> Signup and view all the answers

    What is the expected result when performing replica plating with a tetracycline plate followed by an ampicillin plate?

    <p>Desired colonies will be those that do NOT grow on the ampicillin plate.</p> Signup and view all the answers

    How do pUC plasmids facilitate the screening of colonies with foreign DNA?

    <p>They contain an ampicillin resistance gene and an indicator for b-galactosidase.</p> Signup and view all the answers

    What is a key characteristic of directional cloning?

    <p>It utilizes two different restriction enzymes to create sticky ends.</p> Signup and view all the answers

    Which of the following statements is true regarding competent cells?

    <p>They can take up plasmids and express foreign DNA.</p> Signup and view all the answers

    What role does the sterile velvet transfer tool play in replica plating?

    <p>It transfers clone copies from one plate to another.</p> Signup and view all the answers

    What feature distinguishes blue colonies from colorless colonies on X-gal media?

    <p>Blue colonies have an intact b-galactosidase enzyme, colorless do not.</p> Signup and view all the answers

    In transformant screening, what happens to cells that do not have both plasmid and foreign DNA when exposed to ampicillin?

    <p>They die and do not grow.</p> Signup and view all the answers

    When cloning a gene into a plasmid, why is it important to cut the plasmid with two restriction enzymes?

    <p>To prevent vector re-ligation.</p> Signup and view all the answers

    What is the main function of the Klenow fragment in DNA synthesis?

    <p>It synthesizes DNA but lacks 5′ to 3′ exonuclease activity.</p> Signup and view all the answers

    Which activity does reverse transcriptase lack?

    <p>Proofreading ability</p> Signup and view all the answers

    What is the purpose of making bacterial cells competent?

    <p>To enable them to incorporate foreign DNA.</p> Signup and view all the answers

    What method is traditionally used for bacterial transformation to make cell membranes permeable?

    <p>Heat shock with salt solution</p> Signup and view all the answers

    Which step is NOT part of the six steps of recombinant DNA technology?

    <p>Replication</p> Signup and view all the answers

    What is the primary role of the second strand synthesis in cDNA synthesis?

    <p>To facilitate cDNA formation.</p> Signup and view all the answers

    How are competent E. coli cells typically made?

    <p>By using a heat shock with calcium chloride.</p> Signup and view all the answers

    What is the purpose of replica plating in recombinant DNA technology?

    <p>To transfer colonies onto new media for further analysis.</p> Signup and view all the answers

    What is a feature of plasmids commonly used in recombinant DNA technology?

    <p>They replicate independently of chromosomal DNA.</p> Signup and view all the answers

    Which component is essential for reverse transcriptase to initiate cDNA synthesis?

    <p>RNA template</p> Signup and view all the answers

    What is the goal of cloning cells with a gene of interest?

    <p>To generate copies of a gene</p> Signup and view all the answers

    What type of protein product is typically associated with genetic modifications in plants?

    <p>Genes for pest resistance</p> Signup and view all the answers

    Which of the following best describes the function of a degradative enzyme in bacteria?

    <p>It helps in the breakdown of toxic compounds</p> Signup and view all the answers

    What was a significant finding for Craig Venter regarding his genome?

    <p>A mutation was found that increased his likelihood of heart disease</p> Signup and view all the answers

    What type of proteins are mentioned as products created from modified bacterial cells?

    <p>Enzymes useful in industry</p> Signup and view all the answers

    What implication did the discovery of the 'C' mutation have for Craig Venter?

    <p>It indicated a precaution for future health issues</p> Signup and view all the answers

    What is a common purpose of recombinant DNA technology?

    <p>To produce antibodies for therapies</p> Signup and view all the answers

    What is the primary role of DNA ligase in the process of recombinant DNA technology?

    <p>It repairs single-stranded breaks in DNA.</p> Signup and view all the answers

    Why is it necessary to use calf intestinal phosphatase during the ligation process?

    <p>To prevent the vector DNA from joining with itself.</p> Signup and view all the answers

    What type of bond does DNA ligase form between the 5' phosphate and the 3' hydroxyl during ligation?

    <p>Covalent phosphodiester bond</p> Signup and view all the answers

    What is the result of using a 10:1 ratio of passenger DNA to vector DNA during the ligation process?

    <p>Increased probability of successful ligation of passenger DNA.</p> Signup and view all the answers

    What is the primary energy source used by T4 DNA ligase during the ligation process?

    <p>ATP</p> Signup and view all the answers

    What are recombinant DNA molecules primarily responsible for in molecular biology?

    <p>Combining DNA sequences from different sources</p> Signup and view all the answers

    Which of the following is NOT a step in the six steps of recombinant DNA technology?

    <p>Mutating</p> Signup and view all the answers

    What is the purpose of ligation in recombinant DNA technology?

    <p>To join the vector DNA with the target DNA</p> Signup and view all the answers

    What role do genetically modified organisms play in the application of recombinant DNA technology?

    <p>They enable the large-scale production of substances like insulin</p> Signup and view all the answers

    Which statement accurately describes the source of nucleic acid manipulation technologies that gave rise to recombinant DNA technology?

    <p>Based on diverse sources of nucleic acids from various organisms</p> Signup and view all the answers

    Why was the human gene for insulin placed into bacteria?

    <p>To enable bacteria to produce insulin in large quantities</p> Signup and view all the answers

    Which item is a key feature in the construction of recombinant DNA?

    <p>Presence of a selectable marker</p> Signup and view all the answers

    What is the primary innovation of recombinant DNA technology?

    <p>Combining genetic material from different organisms to create new traits</p> Signup and view all the answers

    What was the outcome of the competition to produce the first complete human genome sequence?

    <p>It was declared a tie and involved intervention from U.S. government.</p> Signup and view all the answers

    What is the main biological role of restriction enzymes in bacteria?

    <p>To prevent the replication of foreign DNA</p> Signup and view all the answers

    Which type of restriction enzyme is most commonly used in recombinant DNA research?

    <p>Type II</p> Signup and view all the answers

    What characteristic is typical of palindromic sequences in DNA?

    <p>The sequences on both strands are identical in sequence</p> Signup and view all the answers

    How do methylases protect host DNA from being cleaved by restriction enzymes?

    <p>By adding methyl groups to specific bases</p> Signup and view all the answers

    Which type of restriction enzyme does not cleave both strands of the DNA?

    <p>Type III</p> Signup and view all the answers

    What is a major feature of Type I restriction enzymes?

    <p>They cleave DNA at locations far away from their recognition sites</p> Signup and view all the answers

    In which scenario would restriction enzymes primarily be utilized?

    <p>In the cloning of genes into plasmids</p> Signup and view all the answers

    What is the composition of Type II restriction enzymes?

    <p>Single-subunit enzymes with distinct functions</p> Signup and view all the answers

    What defines a restriction site in the context of restriction enzymes?

    <p>A specific sequence where cuts are made by the enzymes</p> Signup and view all the answers

    What is a common misconception about restriction enzymes?

    <p>They function randomly without specific targets</p> Signup and view all the answers

    What defines cohesive or 'sticky' ends produced by restriction enzymes?

    <p>They have overhanging single-stranded tails that can base pair with other ends.</p> Signup and view all the answers

    Which part of the naming convention for restriction enzymes indicates the bacterial genus?

    <p>The two-letter abbreviation</p> Signup and view all the answers

    How can different restriction enzymes produce compatible DNA ends?

    <p>If the tails can base-pair even with different recognition sequences.</p> Signup and view all the answers

    Which of the following is NOT a step in the six steps of recombinant DNA technology?

    <p>Amplifying the target gene</p> Signup and view all the answers

    What is the major function of DNA ligase in recombinant DNA technology?

    <p>It joins together different DNA fragments.</p> Signup and view all the answers

    What characterizes isoschizomers in the context of restriction enzymes?

    <p>They recognize the same sequence but are different enzymes.</p> Signup and view all the answers

    Which statement regarding the recognition sites of restriction enzymes is true?

    <p>They consist of palindromic sequences.</p> Signup and view all the answers

    What is the general consequence of using restriction enzymes on DNA?

    <p>It generates fragments with either blunt or sticky ends.</p> Signup and view all the answers

    Why is it important to cut a plasmid with two restriction enzymes in gene cloning?

    <p>To create two compatible ends that prevent self-ligation.</p> Signup and view all the answers

    Which enzyme is essential for covalently linking DNA fragments after restriction enzyme digestion?

    <p>Ligase</p> Signup and view all the answers

    Study Notes

    Recombinant DNA Technology

    • Recombinant DNA technology manipulates genetic material from one organism to another, replicating and expressing it within the new organism.
    • Key figures in its development include Herbert W. Boyer, Stanley N. Cohen, and Paul Berg in 1973. Stanley N. Cohen received a Nobel Prize in 1986 for his work on growth factors.
    • This technology allows for the production of DNA through artificial means. Its ability to change genetic material in living organisms has many potential practical uses, especially in medicine. Many scientists are now investigating recombinant DNA and this field is just beginning to be researched in a concerted effort. 
    • This involves combining DNA from different sources into a single molecule. This is useful in DNA cloning.

    Overview of Recombinant DNA Technology

    • DNA containing a gene of interest is isolated.
    • The plasmid vector is isolated.
    • The desired gene is selected and inserted into the plasmid.
    • The plasmid is taken up by a cell, such as a bacterium.
    • Cells with the gene of interest are cloned.
    • Protein products of a gene are created and harvested.

    Brief History

    • The proposal of the Central Dogma (1957) by Francis Crick
    • Discovery of restriction endonucleases (1970) by Hamilton Smith and Daniel Nathans.
    • Production of the first recombinant DNA using EcoRI enzyme (1972) by Paul Berg.
    • Transforming E. coli with a recombinant plasmid (1973) by Boyer, Cohen, and Chang.
    • First human protein (somatostatin) produced from a transgenic bacterium (1977) by Genentech Inc.
    • Sequencing of the first complete human genome (circa. 2000s) by Craig Venter.

    Basic Concepts of Recombinant DNA Technology

    • The development of laboratory techniques for nucleic acid manipulation in the early 1970s led to the construction of DNA molecules composed of nucleotide sequences from different sources
    • The product of these innovations, recombinant DNA molecules, opened exciting avenues of exploration in molecular biology and genetics creating a new field of study.

    Concept of Recombinant DNA

    • Recombinant DNA is a molecule formed by combining DNA from two different sources. This is synonymous with gene cloning.
    • This creates a new combination of genetic material.
    • An example includes the human gene for insulin being placed in bacteria creating a recombinant organism for mass production of insulin for diabetics. This created a genetically engineered drug in 1986.

    Six steps of Recombinant DNA

    • Isolating (vector and target gene)
    • Cutting (cleavage)
    • Joining (ligation)
    • Transforming
    • Cloning
    • Selecting (Screening)

    Plasmids

    • Small, circular pieces of extrachromosomal DNA found primarily in bacteria, archaea, and eukaryotes.
    • They can replicate independently of the chromosome.
    • They can act as vectors to carry and replicate other pieces of DNA.
    • They are used in clinical, biotechnological, and environmental contexts.
    • Examples of E. coli plasmids include ColEl, F, R100, P1, R6K.
    • Plasmids possess several characteristics: Size ranging from 1 to 4 kb. 

    Plasmids as Vectors

    • Small pieces of DNA used for cloning.
    • Requirements for a vector:  Self-replication (origin of replication) Cloning site (site for restriction endonucleases) Promoter for gene expression in the host A selectable marker (e.g., antibiotic resistance) Appropriate size.

    Cohesive (Sticky) Ends

    • These are products of restriction enzyme digestion.
    • These protruding ends can bind to other ends with the same overhanging sequence.
    • The single-stranded tails can base pair together.

    Restriction Enzymes

    • Molecular "scissors" that cut double-stranded DNA molecules at specific points.
    • Used for making recombinant DNA.
    • Restriction enzymes recognize specific nucleotide sequences (restriction sites) in DNA.
    • Host DNA is protected by methylases which add methyl groups to A or C bases.
    • 3 classes exist (Type I, Type II, and Type III).
    • Type II restriction enzymes are widely useful in recombinant techniques.
    • Restriction enzymes have different cleavage patterns: Blunt ends Cohesive (sticky) ends

    Other Enzymes Used in Recombinant DNA Technology

    • Type II restriction endonucleases (cleave DNA at specific sequences)
    • DNA ligases (join DNA fragments)
    • DNA polymerase I (fills gaps, copies RNA)
    • Reverse transcriptase (copies RNA to DNA)
    • Polynucleotide kinase (adds a phosphate to DNA)
    • Terminal transferase (adds homopolymer tails to DNA)
    • Exonuclease III (removes nucleotides from the ends of DNA)
    • Bacteriophage lambda exonuclease (removes nucleotides from one end of a DNA strand)
    • Alkaline phosphatase (removes terminal phosphates)

    DNA Ligase

    • Enzyme that covalently links DNA molecules.
    • Important for gene cloning.
    • Used to seal gaps in recombinant molecules.
    • Uses energy from ATP to form phosphodiester bonds.

    cDNA Libraries

    • Libraries containing DNA copies (cDNA) of mRNA molecules.
    • Introns are not present, containing only the expressed genes.
    • Essential in studying genes present in mRNA.

    Genomic Libraries

    • Libraries that contain a collection of clones of DNA fragments from one organism that represent the entire genome. 
    • Advantages and disadvantages exist: Advantages = a complete library can show the entire genome, cloning is easy. Disadvantages = expensive, contains some non-essential genes, more time consuming.
    • Important in recognizing particular genes and fragments of DNA within an entire genome.

    Plasmids, Bacteriophages, Cosmids, BACs and YACs as Cloning vectors

    • Plasmids, bacteriophages, and cosmids are cloning vectors used in various applications.
    • Bacterial Artificial Chromosomes (BACs). and Yeast Artificial Chromosomes (YACs) are cloning vectors used in cloning large DNA fragments.

    Expression Vectors

    • Used to produce a protein product.
    • Preferred for high level protein expression to yield desired protein product.

    Methods of Expressing Cloned Genes

    • Techniques that are used in cloning vectors so maximum expression occurs: Strong promoter Insertion Strong termination codon Insertion Adjustment of distance between promoter and cloned gene insertion Insertion of transcription termination sequence Insertion of strong translation initiation sequence

    Bacterial Expression Systems

    • Techniques used to produce eukaryotic proteins in bacteria.
    • Advantages: High level protein production, easy large scale production
    • Disadvantages: Eukaryotic proteins might be recognized as foreign and destroyed, posttranslational modifications may differ.

    Animal Cell Transfection

    • Techniques for introducing foreign DNA into animal cells like in vivo transfections.

    Transposable Elements

    • Mobile genetic elements such as transposons and Controlling elements.
    • Transposable elements can move via DNA or RNA and can be found in eukaryotes and prokaryotes.
    • They can be "selfish DNA" since they only exist to maintain themselves without any useful benefit to the organism.
    • Includes Insertion Sequences (IS elements). DNA transposons, and Retrotransposons (LTR and non-LTR)

    Modes of Genetic Exchange

    • Transformation
    • Phage-mediated transduction
    • Conjugation
    • Transposition

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    Recombinant DNA Technology PDF

    Description

    This quiz explores the fundamentals of recombinant DNA technology, highlighting its development and key figures involved. It focuses on the processes of isolating DNA, using plasmid vectors, and the implications of this groundbreaking technology in medicine. Test your understanding of genetic manipulation and its applications.

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