Molecular Biology and Biotechnology
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Questions and Answers

What is the primary function of a cloning vector?

  • To hybridise DNA strands
  • To carry the gene of interest into a host system (correct)
  • To replicate DNA fragments
  • To synthesise complementary RNA

    • What is the purpose of a multiple cloning site (MCS) in a cloning vector?

  • To create a recombinant plasmid
  • To introduce a restriction enzyme recognition site (correct)
  • To facilitate DNA replication
  • To provide a selectable marker

    • In which type of cell is the host cell selection dependent on the choice of the vector/plasmid?

  • Bacterial cells
  • Yeast cells
  • Mammalian cells
  • All of the above (correct)

    • What is the purpose of heat shock in calcium-rich solution during the transfer of recombinant plasmid into the host cell?

    <p>To make the membrane permeable to the plasmid</p> Signup and view all the answers

    What is the purpose of a genomic library?

    <p>To look at gene structure and compare genomes between organisms</p> Signup and view all the answers

    What is the purpose of a hybridisation probe?

    <p>To identify a specific DNA sequence</p> Signup and view all the answers

    What is the purpose of Southern blotting?

    <p>To detect a specific DNA sequence in a complex mixture</p> Signup and view all the answers

    What is the purpose of microarrays?

    <p>To examine gene expression associated with disease progression</p> Signup and view all the answers

    What is the basis of the polymerase chain reaction (PCR)?

    <p>DNA replication process catalysed by DNA polymerase</p> Signup and view all the answers

    What is the purpose of colony hybridisation?

    <p>To screen bacterial colonies containing recombinant DNA</p> Signup and view all the answers

    What is the primary advantage of PCR in molecular biology?

    <p>It allows for the detection of small amounts of DNA</p> Signup and view all the answers

    Why was the development of PCR a significant breakthrough in molecular biology?

    <p>It enabled the rapid amplification of specific DNA sequences</p> Signup and view all the answers

    What is the primary function of DNA polymerase in PCR?

    <p>To synthesize new strands of DNA complementary to the template</p> Signup and view all the answers

    What is the purpose of the primer in PCR?

    <p>To bind to the single-stranded DNA template</p> Signup and view all the answers

    Why is the use of T.aquaticus DNA polymerase advantageous in PCR?

    <p>It enables the amplification of DNA at high temperatures</p> Signup and view all the answers

    What is the primary difference between Sanger sequencing and Next Generation Sequencing (NGS) technologies?

    <p>NGS uses a single template strand, while Sanger sequencing uses multiple template strands</p> Signup and view all the answers

    What is the primary function of the dideoxynucleotides in Sanger sequencing?

    <p>To terminate DNA synthesis</p> Signup and view all the answers

    What is the primary advantage of NGS technologies over Sanger sequencing?

    <p>NGS allows for the sequencing of thousands of fragments in parallel</p> Signup and view all the answers

    What is the primary requirement for DNA sequencing?

    <p>Many identical copies of the DNA to be sequenced</p> Signup and view all the answers

    Who is credited with the development of the PCR technique?

    <p>Katy Mullis</p> Signup and view all the answers

    What is the primary focus of molecular biology?

    <p>Study of macromolecules and macromolecular mechanisms in living organisms</p> Signup and view all the answers

    What is the main challenge in genetic manipulation?

    <p>Purifying DNA in sufficient amounts from cells or tissues</p> Signup and view all the answers

    What is the enzyme responsible for replicating DNA?

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

    Why is DNA relatively easy to purify?

    <p>Because it is chemically homogeneous</p> Signup and view all the answers

    What is the term for the process of taking a piece of DNA from one place and putting it somewhere it never was?

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

    What is the type of RNA that is most commonly analyzed?

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

    What is the term for the entire set of DNA molecules in the nucleus?

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

    What is the force that holds the two strands of DNA together?

    <p>Hydrogen bonds</p> Signup and view all the answers

    What is the result of exposing DNA to high temperatures?

    <p>The two strands of DNA are separated</p> Signup and view all the answers

    What is the term for the process of producing large amounts of DNA for study?

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

    What is the primary purpose of mechanical disruption in nucleic acid extraction?

    <p>To break open the cell and release its contents</p> Signup and view all the answers

    What is the function of Proteinase K in lysis?

    <p>To break down cellular proteins</p> Signup and view all the answers

    What is the main goal of bacteriophages?

    <p>To produce more bacteriophages by injecting their genome into a host cell</p> Signup and view all the answers

    What is the function of DNA ligase in DNA repair?

    <p>To form phosphodiester bonds between DNA strands</p> Signup and view all the answers

    What is the purpose of restriction enzymes in recombinant DNA technology?

    <p>To cleave DNA at specific sites</p> Signup and view all the answers

    What is the function of gel electrophoresis in DNA analysis?

    <p>To separate DNA molecules based on size and electrical charge</p> Signup and view all the answers

    What is the advantage of using lambda phage vectors over plasmid vectors?

    <p>They can carry larger DNA sequences</p> Signup and view all the answers

    What is the function of expression vectors in recombinant DNA technology?

    <p>To regulate gene expression</p> Signup and view all the answers

    What is the primary advantage of using bacterial plasmids as cloning vectors?

    <p>They are readily obtained and easily introduced into bacterial cells</p> Signup and view all the answers

    What is the characteristic of cloning vectors that makes them useful for DNA manipulation?

    <p>They are self-replicating</p> Signup and view all the answers

    What is the primary reason why DNA is relatively easy to purify?

    <p>Because it is chemically homogeneous</p> Signup and view all the answers

    What is the main function of the enzyme DNA polymerase in DNA replication?

    <p>To replicate DNA</p> Signup and view all the answers

    What is the main purpose of mechanical disruption in nucleic acid extraction?

    <p>To release nucleic acids from cells</p> Signup and view all the answers

    What is the function of restriction enzymes in bacteriophages?

    <p>To defend against bacteriophage infections</p> Signup and view all the answers

    What is the term for the entire set of DNA molecules in the nucleus of a cell?

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

    What is the purpose of DNA ligase in recombinant DNA technology?

    <p>To repair DNA damage</p> Signup and view all the answers

    What is the force that holds the two strands of DNA together?

    <p>Hydrogen bonds</p> Signup and view all the answers

    What is the characteristic of plasmid vectors that makes them useful for DNA manipulation?

    <p>They are self-replicating</p> Signup and view all the answers

    What is the primary focus of molecular biology?

    <p>Studying macromolecules and macromolecular mechanisms</p> Signup and view all the answers

    What is the primary challenge in genetic manipulation?

    <p>All of the above</p> Signup and view all the answers

    What is the primary function of gel electrophoresis in DNA analysis?

    <p>To separate nucleic acids based on size and electrical charge</p> Signup and view all the answers

    What is the purpose of using a lysis buffer in nucleic acid extraction?

    <p>To break open cells</p> Signup and view all the answers

    What is the type of RNA that is most commonly analyzed in molecular biology?

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

    What is the result of exposing DNA to high temperatures?

    <p>The DNA strands separate</p> Signup and view all the answers

    What is the primary advantage of recombinant DNA technology?

    <p>To manipulate DNA molecules</p> Signup and view all the answers

    What is the term for the process of taking a piece of DNA from one place and putting it somewhere it never was?

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

    What is the function of cosmid vectors in DNA manipulation?

    <p>To carry larger DNA sequences</p> Signup and view all the answers

    What is the primary function of plasmid vectors in DNA manipulation?

    <p>To carry foreign DNA into host cells</p> Signup and view all the answers

    What is the primary advantage of molecular biotechnology?

    <p>It allows for the modification of nucleic acids and proteins for applications</p> Signup and view all the answers

    What is the primary advantage of using bacterial plasmids as cloning vectors?

    <p>All of the above</p> Signup and view all the answers

    What is the primary benefit of using PCR in situations where DNA is degraded?

    <p>It creates workable quantities of DNA for research.</p> Signup and view all the answers

    What is the role of DNA polymerase in PCR?

    <p>To synthesize new strands of DNA complementary to the template strand.</p> Signup and view all the answers

    What is the primary advantage of Next Generation Sequencing (NGS) technologies over Sanger sequencing?

    <p>NGS allows for the sequencing of thousands of fragments in parallel.</p> Signup and view all the answers

    What is the purpose of the three steps involved in PCR?

    <p>To denature the DNA, bind primers, and synthesize new DNA strands.</p> Signup and view all the answers

    What is the primary application of DNA sequencing in biology?

    <p>To compare DNA sequences within a species and among separate species.</p> Signup and view all the answers

    What is the primary requirement for DNA sequencing?

    <p>Many identical copies of the DNA to be sequenced.</p> Signup and view all the answers

    What is the primary difference between PCR and DNA sequencing?

    <p>PCR is used for DNA amplification, while DNA sequencing is used for DNA analysis.</p> Signup and view all the answers

    What is the role of the primer in PCR?

    <p>To bind to the single-stranded DNA and initiate DNA synthesis.</p> Signup and view all the answers

    What is the primary benefit of using PCR in research?

    <p>It creates workable quantities of DNA for research.</p> Signup and view all the answers

    What is the primary advantage of using PCR in molecular biology?

    <p>It has revolutionized all aspects of biology.</p> Signup and view all the answers

    What is the primary function of a cloning vector in biotechnology?

    <p>To carry a gene of interest into a host system</p> Signup and view all the answers

    What is the purpose of the multiple cloning site (MCS) in a cloning vector?

    <p>To provide a sequence of tandem restriction endonuclease sites</p> Signup and view all the answers

    What is the effect of high stringency on the binding of a hybridisation probe to its target DNA?

    <p>The probe binds only to perfectly matched targets</p> Signup and view all the answers

    What is the primary difference between a genomic library and a cDNA library?

    <p>A genomic library represents the entire genome, while a cDNA library represents only the expressed genes</p> Signup and view all the answers

    What is the primary purpose of FISH (Fluorescence In Situ Hybridization)?

    <p>To identify and characterise numerical and structural chromosome abnormalities</p> Signup and view all the answers

    What is the function of the restriction enzyme in the Southern blotting technique?

    <p>To cut DNA into smaller fragments</p> Signup and view all the answers

    What is the primary advantage of microarrays over other techniques?

    <p>They enable the examination of gene expression associated with disease progression</p> Signup and view all the answers

    What is the primary function of the heat shock in calcium-rich solution during the transfer of a recombinant plasmid into a host cell?

    <p>To make the membrane permeable to the plasmid</p> Signup and view all the answers

    What is the primary purpose of colony hybridisation?

    <p>To identify bacterial colonies containing recombinant DNA</p> Signup and view all the answers

    What is the primary function of the polymerase chain reaction (PCR) in molecular biology?

    <p>To amplify a specific DNA fragment</p> Signup and view all the answers

    Study Notes

    Cloning Vectors

    • Designed to carry a gene of interest into host systems for expression.
    • Contains a selectable marker (e.g., antibiotic resistance) and unique restriction enzyme recognition sites.
    • Multiple cloning site (MCS) allows for inserting DNA fragments into vectors.

    Cloning Process

    • Cloning refers to creating exact copies of DNA fragments or recombinant plasmids.
    • Host cells (bacteria, yeast, mammalian) are used to reproduce cloned DNA via their own replication mechanism.
    • Selection of host cells is influenced by the choice of vector and intended expression systems.

    DNA Uptake and Transformation Techniques

    • E. coli can uptake DNA when treated with calcium chloride (Mandel & Higa).
    • Heat shock method increases membrane permeability to plasmids; electroporation also enhances uptake via small electric currents.

    DNA Libraries

    • Genomic libraries allow examination of gene structures and genome comparisons.
    • cDNA libraries identify genes encoding proteins specific to tissues.
    • A DNA library represents whole genomes or specific sought-after genes.

    Hybridization Probes

    • Hybridization probes are labeled DNA/RNA fragments used to detect complementary DNA sequences.
    • Stringency determines how well strands must match to bind; controlled by temperature and salt concentration.

    Blotting Techniques

    • Southern blot detects specific DNA sequences by digesting DNA, agarose gel electrophoresis, and transferring DNA to a membrane.
    • Northern blot detects RNA instead of DNA, used for gene expression analysis.
    • In situ hybridization mimics blotting techniques on tissue samples to observe mRNA expression.

    Microarrays

    • Microarrays enable analysis of gene expression and identification of mutations through hybridization principles.
    • Useful for comparing gene expressions in various conditions, such as cancerous vs. normal tissues.

    PCR (Polymerase Chain Reaction)

    • PCR amplifies specific DNA fragments exponentially using DNA polymerase, allowing rapid generation of millions of DNA copies.
    • Involves cycles of denaturation, primer annealing, and extension; essential for various applications in research.

    DNA Sequencing

    • DNA sequencing relies on DNA replication, primarily using Sanger or next-generation sequencing methods.
    • Requires identical DNA copies, DNA polymerase, and modified nucleotides for sequencing accuracy.
    • Next-generation sequencing allows massive parallel sequencing of many DNA fragments.

    Molecular Biology and Biotechnology

    • Molecular biology studies macromolecules and their mechanisms.
    • Molecular biotechnology applies lab techniques to manipulate nucleic acids and proteins.

    Genetic Manipulation

    • Involves isolating, identifying, and characterizing DNA for cloning.
    • Challenges include purifying DNA, manipulating it in the lab, and producing sufficient quantities for study.

    DNA Structure and Composition

    • DNA is a double helix consisting of four nucleotide types linked by phosphodiester bonds.
    • The genome refers to the entirety of DNA within a cell nucleus.
    • DNA polymerase is essential for DNA replication, while RNA can exit the nucleus for protein synthesis.

    Cloning Vectors and Techniques

    • Bacterial plasmids are common cloning vectors due to their easy manipulation and rapid replication.
    • Recombinant DNA technology inserts hybrid DNA from different species into bacterial hosts, showcasing genetic engineering potential.
    • DNA ligase facilitates DNA repair and joining through phosphodiester bond formation.

    Gel Electrophoresis

    • Separated DNA fragments based on size using agarose gels, essential for visualizing nucleic acids.
    • Allows analysis of plasmids and other DNA constructs.

    Summary of Key Concepts

    • DNA is chemically homogenous and relatively easy to purify.
    • RNA analysis focuses primarily on messenger RNA to assess protein-coding gene expression.### Ethanol Precipitation and Nucleic Acid Extraction
    • Ethanol precipitation is a common technique used to purify and concentrate DNA and RNA.
    • Isolation of nucleic acids requires breaking open cells, achievable via mechanical disruption methods.
    • Mechanical disruption techniques include using tissue homogenizers or manual methods like mortar and pestle, especially for plant cells due to rigid cell walls.
    • Lysis uses detergents and enzymes (e.g., Proteinase K) to release DNA and dissolve cellular proteins, often initiated with a lysis buffer.

    Recombinant DNA Technology

    • Involves joining DNA from different species and inserting it into a host cell, typically bacteria.
    • Researchers from UC San Francisco and Stanford employed restriction enzymes to cut specific DNA segments, allowing fusion of DNA from various species.
    • Restriction enzymes (restriction endonucleases) cleave DNA at defined sites, enabling these fusions.

    Bacteriophages and Defense Mechanisms

    • Bacteriophages, viruses that infect bacteria, reproduce by injecting their genome into a bacterial host.
    • Some bacterial strains resist bacteriophage infections, revealing a defense mechanism involving restriction endonucleases that protect against viral DNA.

    DNA Ligase and Cloning

    • DNA ligase repairs DNA by forming phosphodiester bonds between DNA strands, first purified in 1967.
    • Cloning vectors, often plasmids, facilitate the insertion of foreign DNA into host genomes, resulting in transgenic organisms.
    • Plasmids can be modified to include restriction sites and marker genes, allowing for effective cloning.

    Gel Electrophoresis

    • Gel electrophoresis separates nucleic acid fragments by size and charge using an agarose gel.
    • This technique is instrumental in visualizing DNA fragments post-restriction enzyme digestion.

    PCR (Polymerase Chain Reaction)

    • Developed by Katy Mullis in 1985, PCR amplifies DNA from minimal quantities to millions of copies.
    • The process relies on DNA polymerase synthesizing complementary strands based on a known template, following three steps: denaturation, primer annealing, and extension.
    • Taq polymerase, originating from Thermus aquaticus, enables high-temperature reactions without needing to add fresh enzymes after each cycle.

    DNA Sequencing

    • DNA sequencing techniques, such as Sanger (chain-termination) sequencing, rely on DNA polymerases and require large quantities of identical DNA.
    • Sequencing requires temperature-controlled cycling and the use of chain-terminating nucleotides for effective strand synthesis.
    • Next-generation sequencing immobilizes and amplifies a single DNA template to sequence thousands of fragments concurrently.

    Historical Context

    • The structure of DNA was established by Watson and Crick in 1953, with subsequent discovery of human chromosome pairs (23 pairs) in 1956.
    • Advances in sequencing and cloning technologies have revolutionized molecular biology, enhancing the ability to manipulate and study genetic material.

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    Description

    This quiz covers the basics of molecular biology, including the study of macromolecules and mechanisms in living things. It also explores molecular biotechnology, including the use of laboratory techniques to modify nucleic acids and proteins. Test your knowledge of genetic manipulation, DNA purification, and more.

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