Recombinant DNA Technology and Gene Cloning
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What is the primary purpose of recombinant DNA technology?

  • To produce RNA instead of DNA for genetic studies
  • To create DNA sequences that occur naturally in the genome
  • To directly modify the genome of an organism without cloning
  • To amplify a foreign DNA molecule using a host cell (correct)

    • Which of the following accurately describes the function of a vector in gene cloning?

  • To transport the cloned gene into a host cell (correct)
  • To directly edit the genome of an organism
  • To prevent replication of foreign DNA
  • To delete previous DNA sequences from the genome

    • What is the first step in the basics of gene cloning?

  • The vector multiplies within the host cell
  • The DNA fragment is inserted into a vector (correct)
  • The foreign DNA molecule is introduced into the host cell
  • The clone of identical cells is produced

    • What happens to the recombinant DNA molecule when the host cell divides?

    <p>Copies are passed to the progeny cells</p> Signup and view all the answers

    Which statement best describes recombinant DNA (rDNA)?

    <p>It consists of DNA fragments from multiple sources.</p> Signup and view all the answers

    What is the alternative to gene cloning mentioned in the content?

    <p>Polymerase Chain Reaction (PCR)</p> Signup and view all the answers

    Which type of living cell is predominantly used as a host cell in gene cloning?

    <p>Bacterial cells</p> Signup and view all the answers

    Which of the following statements is true about recombinant DNA sequences?

    <p>They can be created using synthetic DNA methods.</p> Signup and view all the answers

    What is a key characteristic of an effective cloning vector?

    <p>It must have a gene for antibiotic resistance.</p> Signup and view all the answers

    Which property is NOT essential for a vector used in cloning?

    <p>Must be capable of integrating into the bacterial chromosome.</p> Signup and view all the answers

    What is the desired copy number for plasmids in a bacterial cell?

    <p>High copy number is desired for maximum yield.</p> Signup and view all the answers

    Why are plasmids commonly used in genetic engineering?

    <p>They can replicate independently of the bacterial chromosomes.</p> Signup and view all the answers

    What happens to large DNA molecules during purification?

    <p>They break down during purification.</p> Signup and view all the answers

    Which of the following is true about bacteriophages used for cloning?

    <p>They consist of a simple structure with a DNA molecule and genes.</p> Signup and view all the answers

    What is the ideal size for plasmids to be effectively used in molecular cloning?

    <p>Under 10 kb.</p> Signup and view all the answers

    What role do selective markers play in plasmid cloning?

    <p>They help in the selection of cells containing the plasmid.</p> Signup and view all the answers

    What is the primary function of Type II restriction endonucleases?

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

    What distinguishes blunt ends from sticky ends created by restriction endonucleases?

    <p>Blunt ends result from cuts in the middle of the recognition sequence</p> Signup and view all the answers

    Which of the following types of vectors is NOT used for cloning large DNA fragments?

    <p>Single-stranded DNA vectors</p> Signup and view all the answers

    What characteristic of recognition sequences makes Type II restriction endonucleases predictable in their cutting pattern?

    <p>They exhibit twofold symmetry or palindromic structure</p> Signup and view all the answers

    What is a key reason for needing to cleave both the vector and the DNA to be cloned?

    <p>To create compatible ends for efficient insertion</p> Signup and view all the answers

    Which example of a restriction enzyme specifically cuts at the hexanucleotide CGATCG?

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

    Which of the following is a characteristic of degenerate recognition sequences in restriction endonucleases?

    <p>They cut at multiple related sites</p> Signup and view all the answers

    In the context of cloning, what is a primary advantage of using cosmids as vectors?

    <p>They are effective for cloning very large DNA fragments</p> Signup and view all the answers

    What are sticky ends in DNA fragments?

    <p>Single-stranded overhangs that result from staggered cuts</p> Signup and view all the answers

    What role does DNA ligase play in the ligation process?

    <p>It joins together individual DNA molecules</p> Signup and view all the answers

    Why is ligation of sticky ends more efficient than that of blunt ends?

    <p>Sticky ends can base pair through hydrogen bonding</p> Signup and view all the answers

    What is the primary purpose of transformation in genetic engineering?

    <p>To introduce DNA into bacterial cells</p> Signup and view all the answers

    What must bacteria undergo to efficiently take up DNA during transformation?

    <p>Chemical or physical treatment</p> Signup and view all the answers

    What does 'competent cells' refer to in the context of transformation?

    <p>Bacterial cells that can take up plasmid DNA efficiently</p> Signup and view all the answers

    What is a disadvantage of the transformation process?

    <p>It is difficult to distinguish transformed cells from non-transformed ones</p> Signup and view all the answers

    Which of the following best describes the effect of using restriction endonucleases with different recognition sequences?

    <p>They can create different cuts leading to the same sticky ends</p> Signup and view all the answers

    What is the reason E.coli cells containing the plasmid pBR322 are resistant to ampicillin?

    <p>pBR322 carries a gene that codes for a b-lactamase enzyme.</p> Signup and view all the answers

    What characteristic distinguishes transformants from non-transformants in E.coli after a transformation experiment?

    <p>Transformants are capable of forming colonies in the presence of ampicillin and tetracycline.</p> Signup and view all the answers

    How can recombinants be identified when using cloning vectors like pBR322?

    <p>The insertion of DNA fragments deactivates one of the original genes.</p> Signup and view all the answers

    Which statement accurately describes the function of the BamHI enzyme in relation to pBR322?

    <p>BamHI disrupts one of the antibiotic resistance genes in the plasmid.</p> Signup and view all the answers

    After transformation with recombinant pBR322, what type of resistance do the cells exhibit?

    <p>Ampicillin resistance only.</p> Signup and view all the answers

    What can be inferred about the majority of colonies that form on the ampicillin medium after transformation?

    <p>Most colonies contain the normal, self-ligated plasmid.</p> Signup and view all the answers

    What is the effect of insertional inactivation on a cloned gene in pBR322?

    <p>The gene cannot be expressed, leading to antibiotic sensitivity.</p> Signup and view all the answers

    What happens to untransformed E.coli cells when plated on ampicillin medium?

    <p>They remain suspended in the medium and do not form colonies.</p> Signup and view all the answers

    What characteristic can be used to identify recombinants when colonies are grown on tetracycline agar?

    <p>Recombinants do not grow and normal colonies regrow.</p> Signup and view all the answers

    Which feature of pUC8 is crucial for recognizing recombinants?

    <p>Incapacity to synthesize b-galactosidase.</p> Signup and view all the answers

    What color do non-recombinant colonies appear when X-gal is added to the agar plate?

    <p>Deep blue</p> Signup and view all the answers

    What is the role of IPTG in the Lac selection screening process?

    <p>To induce the expression of the lacZ' gene.</p> Signup and view all the answers

    What is the purpose of cloning in recombinant DNA technology?

    <p>It enables the production of many recombinant DNA molecules.</p> Signup and view all the answers

    What happens to colonies that harbor a normal pUC8 plasmid within the Lac selection system?

    <p>They are ampicillin resistant and colored blue.</p> Signup and view all the answers

    In transfection, what type of genetic material is inserted into mammalian cells?

    <p>DNA, double-stranded RNA, or plasmids.</p> Signup and view all the answers

    What are recombinants in the context of cloning experiments using the plasmid pUC8?

    <p>Cells that lack the lacZ' gene.</p> Signup and view all the answers

    Study Notes

    Recombinant DNA Technology

    • Recombinant DNA is a piece of DNA created by inserting a DNA fragment from one organism into the replicating DNA of another.
    • It involves combining at least two DNA molecules.
    • Recombinant DNA (rDNA) molecules are formed by laboratory methods of genetic recombination, such as molecular cloning, to combine genetic material from multiple sources.
    • These DNA sequences are not naturally found in a genome, created via genetic engineering.
    • rDNA sequences can come from any organism.
    • Recombinant DNA technology and synthetic DNA allow the creation of any DNA sequence and its introduction into living organisms.
    • Recombinant DNA technology and genetic engineering center around gene cloning.

    Gene Cloning

    • Introducing a foreign DNA molecule into a replicating cell permits cloning or amplification of that DNA.
    • This produces many identical copies of the DNA of interest.
    • PCR is an alternative to gene cloning.

    Basic Steps of Gene Cloning

    • Step 1: Isolate the DNA fragment containing the gene to be cloned and insert it into a circular DNA molecule, called a vector, to make recombinant DNA.
    • Step 2: The vector transports the gene into a host cell (usually a bacterium).
    • Step 3: Inside the host cell, the vector multiplies, creating numerous copies of the gene and itself.
    • Step 4: When the host cell divides, the recombinant DNA is passed on to the offspring and replicates further.
    • Step 5: After many cell divisions, a colony or clone of identical host cells is developed, and each cell in the clone will contain numerous copies of the recombinant DNA. The gene carried by the recombinant molecule is now said to be cloned.

    Vectors for Gene Cloning

    • A vector is a DNA molecule to which the fragment of DNA to be cloned is attached.
    • Common vectors include plasmids and viruses.

    Essential Properties of a Vector

    • Vectors must be able to replicate within the host cells.
    • They must have the ability for autonomous replication in the host cell (many copies).
    • Ideally, vectors should be relatively small (less than 10 kb) to prevent degradation during purification.
    • Vectors should contain at least one specific nucleotide sequence recognizable by restriction endonucleases.
    • It must have at least one gene that controls the selection of the vector (like antibiotic resistance genes).

    Plasmids

    • Plasmids are circular, double-stranded DNA molecules that exist independently within bacterial cells.
    • Plasmids often contain one or more genes that are responsible for specific characteristics of the host bacterium. (Example: antibiotic resistance genes).
    • Plasmids often have an origin of replication, enabling them to replicate independently of the host chromosome.
    • In the lab, antibiotic resistance can often be used as a marker to identify which bacteria contain the specific plasmid.

    Size and Copy Number of Plasmids

    • The ideal size for a plasmid is less than 10 kb.
    • High copy number of the plasmids is favored (high number of plasmid molecules per bacterial cell). A high copy number is desired to obtain large quantities of the recombinant DNA.
    • Low copy number plasmids might be preferable in specific conditions (e.g., toxin production from the cloned gene).

    Bacteriophages

    • Bacteriophages, or phages, are viruses that specifically infect bacteria.
    • They are simple in structure, consisting of a DNA molecule embedded within a protein coat (capsid).

    Bacteriophage Lambda as a Cloning Vector

    • Bacteriophage lambda can be modified to serve as a vector for cloning to carry large DNA fragments (10–23 kb).

    The General Pattern of Infection of a Bacterial Cell by a Bacteriophage

    • The phage attaches to the bacterium and injects its DNA.
    • The phage DNA molecule is replicated.
    • Capsid components are synthesized, phage particles are assembled, and released from the host cell.

    Other Vectors

    • Naturally occurring viruses that infect mammalian cells, like retroviruses, can be used as vectors to clone large DNA fragments.
    • Artificial constructs, such as cosmids, provide alternative cloning vectors.
    • Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are other vectors that can accommodate large DNA fragments.

    Cloning Vectors and Their Insert Capacities

    • Tables present various cloning vector systems and their respective insert capacities, outlining compatibility with host cells and DNA fragment sizes.

    Enzymes for Cutting DNA - Restriction Endonucleases

    • DNA molecules need to be precisely cut.
    • Restriction enzymes cut DNA molecules at specific positions.
    • Each particular restriction enzyme only recognizes one specific sequence.
    • Enzymes cut DNA at a particular nucleotide sequences, termed the "recognition sequence"
    • Some recognition sequences are palindromes.

    Type II Restriction Endonucleases

    • Type II restriction endonucleases cut DNA at specific nucleotide sequences.
    • Each enzyme has a specific recognition sequence at which it cuts a DNA molecule, but nowhere else.

    A Palindrome

    • A palindrome reads the same forward and backward between the 5' and 3' directions

    Type II Restriction Endonucleases Examples

    • Restriction enzymes recognize specific hexanucleotide sequences, such as CGATCG (by PvuII).
    • Some recognize 4, 5, 8, or more nucleotides and recognize a family of related sequences.
    • Specific examples for recognition sequences (and enzymes) are shown in a table.

    Blunt Ends and Sticky Ends

    • Certain restriction enzymes make a straight cut in the DNA (blunt ends), while others generate staggered cuts, creating overhangs (sticky ends).
    • Blunt ends are simpler to work with, but sticky ends facilitate ligation.

    Formation of Recombinant DNA from Restriction Fragments with "Sticky" Ends

    • "Sticky" ends (overhangs) facilitate the ligation process, as complementary bases pair to join fragments.

    Ligation - Joining DNA Molecules Together

    • Ligation involves joining DNA fragments using DNA ligase.
    • DNA ligase catalyzes this reaction, joining individual DNA molecules or the ends of the same molecule.
    • The reaction joins phosphodiester bonds.

    Ligation of Blunt-Ended and Sticky-Ended Molecules

    • Ligation process diagrams represent both blunt-ended and sticky-ended molecule ligations, showcasing differences clearly.

    Sticky Ends Increase the Efficiency of Ligation

    • Sticky ends, with their complementary sequences, increase the efficiency of ligation, as they allow the fragments to align correctly. Ligation with blunt ends is less efficient, needing random collisions.

    Introduction of DNA into Living Cells

    • Transformation is the uptake of DNA by bacterial cells under certain circumstances and conditions.
    • Bacteria need to be modified, or made competent, to efficiently take up DNA.

    Transformation

    • Not all types of bacteria are equally as efficient to take up DNA under normal conditions.
    • Bacteria require modification in the lab to enhance their DNA uptake abilities.

    Selection for Transformed Cells

    • It is important to distinguish cells that have taken up a plasmid from those that have not.
    • This differentiation is usually done by using antibiotic resistance or by using a special enzyme screening mechanism.

    Recombinant Selection with pBR322-Insertional Inactivation of an Antibiotic Resistance Gene

    • Recombinant selection with pBR322 usually involves inactivation of an antibiotic resistance gene by insertion of foreign DNA.

    After Transformation

    • Transformants and non-transformants are easily differentiated and separated from each other.

    To Identify Recombinants

    • Replica plating technique is used to identify cells that do or do not grow after incubation on a certain medium.
    • Cells that do not grow are the ones where the DNA of interest has been inserted into the inactivation gene of the cloning vector.

    Insertional Inactivation

    • Insertional inactivation is usually applied in gene cloning to distinguish between recombinants and non-recombinants.

    Insertional inactivation does not always involve antibiotic resistance - Lac Selection/Blue-White Screen

    • Insertional inactivation can also be used with the Lac Selection system, a method that uses a gene that codes for part of the enzyme b-galactosidase, which is used to distinguish recombinants from non-recombinants.

    B-galactosidase

    • The enzyme b-galactosidase breaks down X-gal into a blue product, while a non-recombinant, functional lacZ gene will lead to a blue color.
    • A recombinant will not be able to produce B-galactosidase, leading to a white color.

    Transfection

    • Transfection is a process in which genetic material such as DNA and/or RNA is introduced into mammalian cells.

    Transfection Methods

    • There are different methods for transfecting mammalian cells, like liposome transfection, viral transduction, electroporation, optoporation, and microinjection.

    Cloning Serves Two Main Purposes

    • Cloning enables the production of numerous recombinant DNA molecules from a limited starting material.
    • Cloning provides a pure sample of an individual gene, separated from other genes.

    Purification

    • Cloning techniques allow for the separation and purification of specific genes from a complex sample of DNA.

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    Explore the principles of recombinant DNA technology and the process of gene cloning. This quiz covers how DNA fragments from different organisms are combined, the methods used in molecular cloning, and the significance of PCR in DNA amplification. Test your knowledge on these essential techniques in genetic engineering.

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