Genetic Engineering Overview
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Questions and Answers

What is the primary purpose of using terminal deoxynucleotidyl transferase in recombinant DNA formation?

  • To amplify the DNA
  • To remove unwanted nucleotide sequences
  • To add complementary tails to DNA fragments (correct)
  • To cut DNA into fragments

    • Blunt end ligation has a high efficiency by itself without any further modifications.

    False

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

    To covalently link DNA fragments together.

    Calf intestinal phosphatase is used to remove the _______ from linearized vector DNA.

    <p>5′ phosphate</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Sticky ends = Allow for more efficient ligation due to complementary base pairing Blunt ends = Have no overhanging nucleotides DNA ligase = Enzyme that covalently bonds DNA fragments Restriction endonucleases = Enzymes that cut DNA at specific sequences</p> Signup and view all the answers

    Which of the following statements is true about DNA ligase?

    <p>It forms phosphodiester bonds between DNA fragments.</p> Signup and view all the answers

    Restriction endonucleases can create both sticky and blunt ends.

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

    What is the primary function of DNA ligase in molecular biology?

    <p>To covalently bond DNA fragments together by forming phosphodiester bonds.</p> Signup and view all the answers

    DNA fragments can anneal to form recombinant molecules through ________ bonding.

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

    Match the type of DNA ends with their characteristics:

    <p>Sticky ends = Have unpaired bases that can easily anneal with complementary sequences Blunt ends = Have no unpaired bases and are straight cut Cohesive ends = Another term for sticky ends, promoting easier joining Phosphodiester bonds = Covalent bonds formed by ligase between DNA strands</p> Signup and view all the answers

    What type of enzyme requires ATP for its function?

    <p>DNA ligase</p> Signup and view all the answers

    T4 DNA ligase exclusively ligates sticky-ended fragments.

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

    Why are higher concentrations of T4 DNA ligase usually necessary for ligating blunt ends?

    <p>Because the reaction is less efficient with blunt ends compared to sticky ends.</p> Signup and view all the answers

    What is the main function of DNA ligase in the process of recombinant DNA formation?

    <p>To covalently link DNA fragments together</p> Signup and view all the answers

    Restriction endonucleases create sticky ends on DNA fragments.

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

    What is the role of BamHI in the recombinant DNA process?

    <p>BamHI is a restriction endonuclease that cleaves DNA at specific sequences to create sticky ends.</p> Signup and view all the answers

    The process that covalently links two DNA fragments together is called __________.

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

    Match the following terms with their descriptions:

    <p>Sticky ends = Single-stranded overhangs that allow DNA fragments to bind Blunt ends = Straight cut ends with no overhangs Restriction enzymes = Proteins that cut DNA at specific sequences DNA ligase = Enzyme that links together DNA fragments</p> Signup and view all the answers

    Which mechanism of DNA cleavage is used to generate sticky ends?

    <p>Cuts made by restriction endonucleases like BamHI</p> Signup and view all the answers

    Blunt ends are more favorable for DNA ligation than sticky ends.

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

    What does terminal deoxynucleotidyl transferase do?

    <p>It adds deoxynucleotides to the 3′OH ends of DNA molecules.</p> Signup and view all the answers

    The enzyme __________ can create sticky ends for the purpose of DNA recombination.

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

    Match the following enzymes with their main functions:

    <p>BamHI = Cuts DNA to create sticky ends T4 DNA Ligase = Links DNA fragments together DNA Polymerase III = Replicates DNA strands Terminal Transferase = Adds nucleotides to 3' ends of DNA</p> Signup and view all the answers

    What are the two types of cuts that type II restriction endonucleases can produce?

    <p>Blunt ends and sticky ends</p> Signup and view all the answers

    EcoRI is an enzyme that produces blunt ends when it cleaves DNA.

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

    What is the primary function of a type II restriction endonuclease?

    <p>To cleave DNA at specific recognition sequences.</p> Signup and view all the answers

    Type II restriction endonucleases typically recognize symmetric DNA sequences of ______ base pairs.

    <p>4-8</p> Signup and view all the answers

    Match the restriction endonuclease with its cutting style:

    <p>EcoRI = Creates sticky ends SmaI = Creates blunt ends HindIII = Creates sticky ends NotI = Creates sticky ends</p> Signup and view all the answers

    Which of the following statements is true about type II restriction endonucleases?

    <p>They require Mg2+ as a cofactor.</p> Signup and view all the answers

    Restriction endonucleases only cut double-stranded DNA.

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

    What is generated at the ends of DNA after cleavage by type II restriction endonucleases?

    <p>Free 5′-phosphate and 3′-OH ends.</p> Signup and view all the answers

    The symmetrical recognition sequences of restriction enzymes are also referred to as ______.

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

    Why are sticky ends beneficial in molecular biology applications?

    <p>They allow for easier ligation of DNA fragments.</p> Signup and view all the answers

    Study Notes

    Genetic Engineering

    • Genetic engineering is the artificial manipulation of an organism's nucleic acid content, differing from genetic recombination, which is a natural process.
    • Recombinant DNA (rDNA) is artificially created by combining DNA sequences from different sources.
    • Genetically modified organisms (GMOs) have had their genes artificially altered.

    Genetic Engineering Steps

    • Cleave vector with restriction enzyme: A restriction enzyme cuts a plasmid vector at specific restriction sites.
    • Cleave foreign DNA: A restriction enzyme cuts foreign DNA at matching restriction sites.
    • Mix vector and DNA fragment: The vector and DNA fragment are mixed to encourage base pairing between complementary sequences.
    • Treat with DNA ligase: DNA ligase covalently joins the DNA pieces.
    • Recombinant vector: The resulting recombinant vector carries the gene of interest.

    Recombinant DNA Technology Applications

    • Studying gene arrangement, expression, and regulation.
    • Modifying gene expression to enhance or suppress a specific product.
    • Creating multiple copies of a nucleic acid segment.
    • Creating organisms with altered characteristics (transgenic organisms).
    • Using transgenic organisms as cell factories for commercial purposes.

    Enzymes Used in Cloning-Restriction Endonucleases

    • Restriction enzymes (REs) are endonucleases that cut DNA at specific recognition sites.
    • Restriction sites are short, specific nucleotide sequences.
    • REs are bacterial in origin.
    • Three major classes of REs exist, based on their recognition sequences, cut type, and enzyme structure.
    • Type I and III are not commonly used in cloning due to non-specific cutting sites.
    • Type II enzymes are widely used for cloning because they cut precisely at recognition sites.

    Restriction Endonuclease Nomenclature

    • REs are named based on the organism they are isolated from (e.g., HindIII, EcoRI, BamHI).

    Recognition Sequences for Type II Restriction Endonucleases

    • These enzymes recognize specific palindromic sequences.
    • Some create "sticky ends"—single-stranded overhangs that form hydrogen bonds with complementary sequences.
    • Others create "blunt ends," producing even cuts in the DNA.

    Ligases

    • DNA ligases form covalent bonds between DNA fragments' 5' and 3' ends.
    • T4 DNA ligase is commonly used to connect DNA with sticky or blunt ends.
    • Ligation is the joining of DNA fragments using ligase.

    Terminal Deoxynucleotidyl Transferase

    • This enzyme adds deoxynucleotides to the 3' ends of DNA molecules.

    Phosphatases

    • Removal of 5' phosphate from DNA molecules, preventing self-ligation of the vector.
    • Calf intestinal phosphatase is used.

    Large Fragment of DNA Polymerase I (Klenow Fragment)

    • Cleavage of intact DNA polymerase I produces this 76-kDa enzyme.
    • This enzyme possesses 5' to 3' polymerase and 3' to 5' exonuclease activity.
    • Used in DNA sequencing, filling recessed 3' termini, labeling DNA fragments.

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    Molecular DNA Technology: Genetic Engineering Notes PDF

    Description

    This quiz explores the fundamental concepts of genetic engineering, including the processes involved in recombinant DNA technology and its applications. Test your knowledge on the steps and significance of genetic modifications in organisms. Perfect for students and enthusiasts in the field of genetics!

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