Molecular Cloning and Biotech Basics
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Questions and Answers

What is the recognition sequence for Dam methylase?

  • ATGC
  • AAGC
  • CTAG
  • GATC (correct)

    • What type of ends do XbaI generate when cut at its recognition site?

  • Non-compatible ends
  • Blunt ends
  • Sticky ends with overhang (correct)
  • Compatible sticky ends

    • What is required during the ligation process after cutting DNA with restriction enzymes?

  • Nucleotide triphosphates
  • Phage DNA ligase and ATP (correct)
  • DNA polymerase
  • RNA ligase

    • Which component is essential for the cloning efficiency of a plasmid?

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

    Why are sticky ends more efficient for cloning than blunt ends?

    <p>They can form hydrogen bonds more easily.</p> Signup and view all the answers

    What does the presence of blue colonies indicate in bacterial transformation experiments?

    <p>Functional LacZ is produced.</p> Signup and view all the answers

    What is the consequence of inserting DNA into the plasmid during cloning?

    <p>Functional LacZ is not synthesized.</p> Signup and view all the answers

    If your cloning experiment shows no white colonies, what is the most likely reason?

    <p>Your plasmid has no inserts.</p> Signup and view all the answers

    In the context of E.coli expression vectors, which element is crucial for driving expression of a cloned gene?

    <p>A strong promoter, like T7 phage gene promoter.</p> Signup and view all the answers

    What role does a His-tag serve in protein expression systems?

    <p>To facilitate protein purification.</p> Signup and view all the answers

    What is the significance of human insulin in the context of biotechnology?

    <p>It was the first recombinant pharmaceutical approved by the FDA in the U.S.</p> Signup and view all the answers

    Which of the following accurately describes restriction endonucleases?

    <p>They cut only double-strand DNA targets.</p> Signup and view all the answers

    What type of genes do naturally occurring plasmids typically harbor?

    <p>Non-essential genes that may enhance survival.</p> Signup and view all the answers

    What is the primary function of EcoRI restriction endonuclease?

    <p>To cut double-stranded DNA and leave sticky ends.</p> Signup and view all the answers

    What happens to the activity of restriction endonucleases when DNA is methylated?

    <p>Their activity is inhibited.</p> Signup and view all the answers

    What is the role of the lacZ gene in the context of blue-white screening?

    <p>It encodes the enzyme beta-galactosidase.</p> Signup and view all the answers

    What function does IPTG serve in the lac operon?

    <p>It is a gratuitous inducer that mimics lactose.</p> Signup and view all the answers

    How does blue-white screening differentiate between colonies?

    <p>Through the expression of beta-galactosidase.</p> Signup and view all the answers

    What happens to colonies containing a functional lacZ gene when X-gal is present?

    <p>They form blue colonies.</p> Signup and view all the answers

    What effect does placing an MCS within the lacZ gene have on plasmid function?

    <p>It disrupts the lacZ gene and prevents beta-galactosidase activity.</p> Signup and view all the answers

    What role does IPTG play in the T7 RNA polymerase system?

    <p>It induces the expression of the lac operon.</p> Signup and view all the answers

    Which component is essential for the binding of His-tagged proteins during protein purification?

    <p>Nickel resin</p> Signup and view all the answers

    What is the primary purpose of the Sanger sequencing method?

    <p>To obtain a sequence of a specific gene</p> Signup and view all the answers

    Meta-genomics can be best described as:

    <p>The analysis of all genes from microbial communities.</p> Signup and view all the answers

    What is the advantage of using Next Generation Sequencing over traditional methods?

    <p>It allows for the sequencing of millions of reads simultaneously.</p> Signup and view all the answers

    Which of the following processes involves the expression of cloned genes?

    <p>Protein expression and purification</p> Signup and view all the answers

    What is the main focus of the CRISPR/Cas gene editing technology?

    <p>Targeted modification of specific genes</p> Signup and view all the answers

    Which statement is true regarding the T7 promoter?

    <p>It is often used to drive gene expression in cloning.</p> Signup and view all the answers

    What does CRISPR/Cas stand for?

    <p>Clustered Regularly Interspaced Short Palindromic Repeats</p> Signup and view all the answers

    How does the CRISPR/Cas system function in bacteria?

    <p>It stores DNA from phages to recognize and defend against future infections.</p> Signup and view all the answers

    What is a key component of the CRISPR/Cas gene editing tool?

    <p>Cas9 endonuclease</p> Signup and view all the answers

    What mutation causes sickle cell anemia?

    <p>Point mutation from glutamic acid to valine</p> Signup and view all the answers

    In gene cloning, what is the purpose of using restriction enzymes?

    <p>To digest a plasmid and clone genes into it</p> Signup and view all the answers

    What can be included in a repair template during gene editing?

    <p>A new desired sequence</p> Signup and view all the answers

    Which of the following features is not a part of a typical plasmid used for cloning?

    <p>RNA transcription site</p> Signup and view all the answers

    What technique is used for studying protein activity in vitro?

    <p>Affinity tagging and purification</p> Signup and view all the answers

    Study Notes

    Molecular Biology I and II

    • Topics covered in Molecular Biology II include cloning, protein expression and purification, DNA sequencing, and CRISPR/Cas gene editing
    • Molecular Biology I topics include recombination and gene deletion, using bacteria to assay mutagens, mutant libraries and transposons, PCR, qPCR, RT-qPCR, gel electrophoresis, hybridization of nucleic acids (Northern and in situ hybridization)

    Gel Electrophoresis

    • Agarose gel is used
    • Takes advantage of the inherent charge of nucleic acids
    • Linear molecules fractionate through the agarose matrix based on size
    • The direction of movement of the molecules depends on the electrode configuration

    DNA Sequencing - Sanger Method

    • Uses dideoxynucleotides (ddNTPs) to stop DNA synthesis at specific points
    • Fluorescently labeled ddNTPs are used to identify the bases
    • Gel electrophoresis separates the DNA fragments by size, revealing the DNA sequence
    • Next-generation sequencing methods provide millions of sequence reads for large-scale sequencing projects

    Nucleic Acid Hybridization

    • Relies on the ability of complementary strands of nucleic acids (NA) to hydrogen bond
    • Used to find a target gene and to determine if a gene is expressed

    Northern Hybridization

    • RNA samples are probed with a gene probe to determine gene expression

    In Situ Hybridization

    • DNA probe is used to find a cell that contains the specific sequence

    Cloning into a Plasmid Vector

    • Foreign DNA is cut with restriction enzymes to create sticky ends
    • A vector is cut with the same restriction enzymes
    • DNA ligase is used to form recombinant molecules
    • Recombinant vectors are introduced into a host

    Naturally Occurring Plasmids

    • Harbor nonessential genes that may enhance survival
    • Have a size range of 1kb to >1 Mb
    • Replicate and segregate independently of the chromosome

    Restriction Enzymes

    • Restriction endonucleases break down sugar-phosphate backbones
    • Exonucleases remove bases from one end of a nucleic acid molecule
    • Restriction endonucleases are found in bacteria and archaea
    • They cut double-stranded DNA.

    EcoRI

    • An example of a restriction endonuclease
    • Produces sticky ends
    • Estimated frequency of genomic DNA cut sites is about one site per 4,096 base pairs

    DNA Methylation

    • Affects RE activity
    • Methylated sequences are less likely to be cut by restriction enzymes

    Xbal

    • An example of a restriction endonuclease found in Xanthomonas badrii

    Cloning into a Plasmid Vector

    • Foreign DNA and plasmid DNA are cut with the same restriction endonucleases
    • Sticky ends are formed and linked up using DNA ligase.

    Compatible DNA Fragments

    • Using restriction enzymes that produce sticky ends to create fragments that can be ligated together to form new DNA molecules
    • This method is preferred over using blunt ends for ligation as more ligation possibilities are available.

    Blue-White Screening

    • Used to identify recombinant plasmids
    • A gene coding for beta-galactosidase is inserted into a plasmid
    • Beta-galactosidase breaks down X-gal to produce a color change

    The lac promoter

    • Is a common promoter for gene expression of many different genes/proteins
    • Includes important proteins like LacZ, LacY, and LacA
    • Uses components such as repressor gene (lacl), promoter, and inducer for regulation

    The Lac Promoter (using IPTG)

    • IPTG is a gratuitous inducer for the lac operon
    • It is similar to lactose but is not metabolized by the cell
    • It is an effective way to induce the lac operon for consistent gene expression

    Essential components of a plasmid for cloning and gene expression

    • Origin of replication (p15A ori)
    • Multiple Cloning Site (MCS)
    • Promoter
    • Selectable marker

    MCS-region with many RE sites

    • Contains many restriction enzyme cut sites
    • Used as a cloning site
    • Example: pUC19 (2686 base pairs) bacterial plasmid

    CRISPR/Cas

    • Clustered Regularly Interspaced Short Palindromic Repeats
    • Bacterial adaptive immunity system
    • Contains a Cas9 endonuclease
    • Used in gene editing by targeting specific DNA sequences

    CRISPR/Cas Gene Editing

    • Use of CRISPR/Cas9 system to cut DNA at a specific location, and potentially to introduce a repair template
    • Includes use of guide RNA that helps the Cas9 enzyme to cut DNA in the correct spot

    Expression Vectors for E. coli

    • Strong promoter (T7 phage gene promoter) to drive expression of the cloned gene
    • Need to use engineered E. coli strain to express T7 RNA polymerase
    • Have a terminator sequence
    • Have an RBS sequence for efficient protein synthesis

    Protein Purification

    • Steps involved in purifying a protein with different methods to purify the correct product from the rest of the mixtures
    • Use of His-tag to bind proteins to nickel resin, helping purify desired proteins

    Sequencing Methods

    • Sanger Sequencing: uses dideoxynucleotides to stop DNA synthesis at specific points, and gel electrophoresis is used to determine sequence based on fragment sizes
    • Next-Generation Sequencing: uses millions of sequence reads in large-scale sequencing projects, with fluorescently labeled nucleotides, and gel electrophoresis
    • Both Sanger and Next-Generation methods are suitable for different applications

    Metagenomics

    • Isolating and sequencing all DNA from a microbial community in its natural environment to see the "big picture"
    • Analyze all DNA from a sample of sea water

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    Molecular Biology II Fall 2024 PDF

    Description

    Test your knowledge on essential concepts in molecular cloning and biotechnology. This quiz covers key topics such as restriction enzymes, cloning efficiency, and plasmid components. Ideal for students and professionals alike, it's a great way to reinforce your understanding of these fundamental biological processes.

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