Cell stuff-8
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Questions and Answers

What distinguishes sticky fragments from blunt fragments in DNA cleavage?

  • Sticky fragments have one strand that is longer or shorter than the complementary strand, while blunt fragments do not. (correct)
  • Blunt fragments can only be produced by certain restriction enzymes.
  • Sticky fragments are cut straight across, while blunt fragments are not.
  • Blunt fragments have complementary strands of equal length, while sticky fragments do not.

    • Which type of gel is appropriate for separating DNA fragments that are between 0.1 and 50 kilobase pairs?

  • Agarose gel (correct)
  • Pulsed-field agarose gel
  • Polyacrylamide gel
  • Nucleotide gel

    • What role does DNA ligase play in the recombinant DNA technique?

  • It separates DNA fragments during electrophoresis.
  • It amplifies and propagates plasmids within bacteria.
  • It cuts specific sequences of DNA to create sticky ends.
  • It fuses two DNA fragments, either with compatible or blunt ends. (correct)

    • How is cDNA created from mRNA in the process of recombinant DNA technology?

    <p>Through a process called reverse transcription with reverse transcriptase.</p> Signup and view all the answers

    What was the first recombinant protein produced using recombinant DNA technology?

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

    What kind of fragments do pulsed-field agarose gels separate?

    <p>Chromosomal fragments ranging from 100 kb to 5 Mb</p> Signup and view all the answers

    Which enzyme is responsible for creating sticky ends in DNA fragments?

    <p>Restriction enzymes</p> Signup and view all the answers

    Which of the following statements is true regarding plasmids used in genetic engineering?

    <p>Plasmids independently propagate within bacteria.</p> Signup and view all the answers

    What modifying function does an expression vector provide in recombinant DNA technology?

    <p>It contains a promoter for enhanced expression of the inserted gene.</p> Signup and view all the answers

    Which of the following is a benefit of using recombinant DNA technology for insulin production?

    <p>It can produce large quantities of identical recombinant proteins.</p> Signup and view all the answers

    What is the primary function of primers in DNA synthesis?

    <p>To provide start points for synthesis</p> Signup and view all the answers

    How does the presence of excess probes affect DNA hybridization?

    <p>It favors probe-hybridization over renaturation.</p> Signup and view all the answers

    What is the role of DNA polymerase during PCR?

    <p>To extend primers in the 5' to 3' direction</p> Signup and view all the answers

    What happens during the first cycle of PCR?

    <p>Single-strands become double-stranded DNA</p> Signup and view all the answers

    What is a characteristic of short tandem repeats (STR) in the human genome?

    <p>They vary in length, providing unique genetic fingerprints.</p> Signup and view all the answers

    What is the necessity for using specific primers in PCR?

    <p>To clone any DNA or RNA fragment accurately</p> Signup and view all the answers

    Which statement describes the cloning process mentioned in the content?

    <p>Is restricted to using known sequences for success</p> Signup and view all the answers

    What is one outcome of temperature cycling in PCR?

    <p>Amplification of a specific stretch of DNA</p> Signup and view all the answers

    What is the theoretical match rate for probes as compared to nucleotides?

    <p>1 in 10^18</p> Signup and view all the answers

    During the second cycle of PCR, what occurs to the number of single-stranded DNA molecules?

    <p>They double from the previous cycle's count.</p> Signup and view all the answers

    What is the primary purpose of using probes in DNA hybridization?

    <p>To detect specific DNA sequences through labeling</p> Signup and view all the answers

    What effect does excess probe concentration have during hybridization?

    <p>Enhances the binding of probes to target DNA</p> Signup and view all the answers

    Why are specific primers essential for PCR?

    <p>They enable the cloning of any DNA or RNA fragment</p> Signup and view all the answers

    How does DNA polymerase contribute to PCR?

    <p>It extends primers in the 5' to 3' direction</p> Signup and view all the answers

    What characterizes the output after two cycles of PCR?

    <p>The number of double-stranded DNA molecules quadruples</p> Signup and view all the answers

    What unique information can be obtained through measuring short tandem repeats (STRs) in genomes?

    <p>A unique fingerprint for each individual genome</p> Signup and view all the answers

    What is a critical factor for effective PCR when targeting an infectious agent's DNA/RNA?

    <p>Knowledge of the sequence information for specific primers</p> Signup and view all the answers

    How is the theoretical match of probes calculated in relation to nucleotides?

    <p>1 in 10^18 nucleotides</p> Signup and view all the answers

    During which phase of PCR do primers attach to single-stranded DNA?

    <p>After cooling during the annealing step</p> Signup and view all the answers

    What is the advantage of restriction-free, cell-free cloning mentioned in the context?

    <p>It is faster than conventional cloning methods</p> Signup and view all the answers

    Which of the following describes the outcome when restriction enzymes are employed in DNA manipulation?

    <p>They generate both blunt and sticky ends depending on the enzyme used.</p> Signup and view all the answers

    What is the primary advantage of using plasmids in recombinant DNA technology?

    <p>They provide an independent replication mechanism in a bacterial host.</p> Signup and view all the answers

    Which characteristic best distinguishes cDNA from genomic DNA?

    <p>cDNA is synthesized from mRNA and lacks introns.</p> Signup and view all the answers

    What does the presence of specific restriction enzyme recognition sequences ensure in DNA manipulation?

    <p>Different enzymes with specific sequences lead to unique fragment patterns.</p> Signup and view all the answers

    In the process of separating DNA fragments by gel electrophoresis, what is a crucial factor that affects the migration of DNA?

    <p>The size of the DNA fragments significantly affects their movement through the gel.</p> Signup and view all the answers

    Which statement accurately describes the use of expression vectors in recombinant DNA technology?

    <p>Expression vectors facilitate the expression of cDNA in bacterial systems.</p> Signup and view all the answers

    What happens during the ligation process in recombinant DNA technology?

    <p>DNA fragments with complementary ends are fused together with the help of ATP.</p> Signup and view all the answers

    What distinguishes the use of pulsed-field agarose gel from standard agarose gel in DNA analysis?

    <p>Pulsed-field agarose gels can separate much larger DNA molecules, such as chromosomes.</p> Signup and view all the answers

    Which of the following is a feature of recombinant insulin production?

    <p>The plasmid containing cDNA is inserted into bacteria to produce insulin.</p> Signup and view all the answers

    Which of the following statements is correct regarding the characteristics of DNA fragments generated by restriction enzymes?

    <p>Each restriction enzyme may produce fragments with unique sizes and ends.</p> Signup and view all the answers

    Study Notes

    Restriction Enzymes

    • Sequence-specific endonucleases that cleave DNA into fragments.
    • Create either blunt or sticky ends.
    • Each enzyme has a unique recognition sequence and creates a specific sticky end.

    DNA Fragment Separation

    • Restriction enzymes cut DNA into fragments of different sizes.
    • Fragments are separated by size using gel electrophoresis.
    • Polyacrylamide gel is used for small fragments (10-1000 bases).
    • Agarose gel is used for larger fragments (0.1 - 50k base pairs).
    • Pulsed-field agarose gel is used for very large fragments (chromosomes, 100 kb - 3.5 Mb).

    Plasmid Vectors

    • Independent, circular DNA molecules that replicate in bacteria.
    • Can be easily separated from chromosomal DNA.
    • Used as vectors for cloning and expressing foreign DNA.

    Recombinant DNA Techniques

    • Involves inserting a desired DNA sequence into a plasmid vector using restriction enzymes and ligase.
    • The recombinant plasmid can be used to produce proteins or study gene function.

    Genomic Library

    • A collection of clones, each containing a different fragment of the genome.
    • DNA fragments are inserted into plasmids and transformed into bacteria.
    • Allows for the study and analysis of entire genomes.

    Producing Proteins by Recombinant DNA

    • cDNA (complementary DNA) is used to create recombinant proteins.
    • cDNA is derived from mRNA and contains only coding sequences.
    • Expression vectors contain promoters that drive gene expression.
    • Cloning cDNA into expression vectors creates bacteria that overexpress the recombinant protein.

    Producing Insulin by Recombinant DNA

    • The first recombinant protein produced.
    • mRNA from the pancreas is transcribed into proinsulin cDNA using reverse transcriptase.
    • The cDNA is inserted into a plasmid and introduced into bacteria.
    • Bacteria produce large amounts of insulin protein.

    Modified Nucleotides

    • Radioactive (32P), fluorescent (many dyes), and chemically tagged (e.g., DIG).

    Probes

    • Short DNA fragments (around 30 nucleotides) used to identify complementary sequences.
    • Often labeled for detection.
    • Used in hybridization experiments to detect specific DNA sequences.

    Primers

    • Short DNA fragments (16-24 nucleotides) used as starting points for DNA synthesis.
    • Used in PCR and other DNA amplification techniques.

    Hybridization of Probes

    • Probes can hybridize to single-stranded DNA.
    • Excess probe favors probe-hybridization over renaturation of original strands.
    • Labeling of probes allows for detection.

    DNA Polymerase

    • Enzyme that synthesizes DNA, extending primers in the 5' to 3' direction.
    • Crucial for DNA replication and PCR.

    PCR (Polymerase Chain Reaction)

    • Amplifies specific DNA sequences using temperature cycling.
    • Denaturation: Heat separates double-stranded DNA into single strands.
    • Annealing: Primers bind to complementary sequences on the single strands.
    • Extension: DNA polymerase extends the primers, copying the DNA sequence.
    • Repeated cycles exponentially amplify the target DNA sequence.

    Applications of PCR

    • Cloning DNA fragments.
    • Diagnosing infections.
    • Detecting genetic mutations.
    • Forensic science.
    • Establishing paternity.

    Short Tandem Repeats (STRs)

    • Variable length of repeats in the genome.
    • Measured by PCR to determine the length of the repeats.
    • Used in forensic science to create individual DNA fingerprints.

    Restriction Enzymes and DNA Fragmentation

    • Restriction enzymes are proteins that cut DNA at specific sequences, creating fragments.
    • These fragments can have blunt ends (both strands cut straight) or sticky ends (one strand longer than the other).
    • The sticky ends are complementary, allowing fragments to be joined.
    • Over 100 known restriction enzymes with different recognition sequences are available.

    Separating DNA Fragments

    • After restriction enzyme digestion, DNA fragments can be separated by size using gel electrophoresis.
    • Smaller fragments migrate faster through the gel.
    • Different gels are used depending on fragment size:
      • Polyacrylamide gels for nucleotides (10 - 1000 bases).
      • Agarose gels for larger DNA fragments (0.1 - 50k base pairs).
      • Pulsed-field agarose gels for chromosomes (100kb - 3.5Mb).

    DNA Ligation

    • DNA ligase joins DNA fragments with compatible ends, including blunt ends.
    • The enzyme requires ATP to catalyze the reaction.

    Plasmid Vectors

    • Plasmids are circular DNA molecules that can replicate independently in bacteria.
    • They can be easily separated from chromosomal DNA.
    • Plasmids are used in recombinant DNA techniques.

    Recombinant DNA Technology

    • Involves inserting a new DNA sequence into a plasmid vector.
    • This is achieved by cleaving both the plasmid and the desired DNA sequence with restriction enzymes.
    • The resulting fragments are then joined using DNA ligase.

    Genomic Libraries

    • A collection of DNA segments representing the entire genome, cloned into separate bacterial cells.
    • Each bacterial clone harbors a specific DNA fragment from the original genome.
    • This allows for the isolation and analysis of individual genes.

    Recombinant Protein Production

    • cDNA, synthesized from mRNA, lacks introns and provides a continuous coding sequence.
    • An expression vector containing a strong promoter is used to express the cloned cDNA in bacteria.
    • This results in the production of recombinant proteins.

    Insulin Production Using Recombinant DNA Technology

    • The first recombinant protein produced.
    • mRNA from the pancreas is converted to cDNA with reverse transcriptase.
    • The cDNA is then inserted into a plasmid and propagated in bacteria.
    • The bacteria produce insulin in significant quantities.

    Modified Nucleotides

    • Fluorescent and radioactive nucleotides are used for labelling and detection in various biological experiments.
    • Chemically tagged nucleotides, such as DIG, are also used for specific applications.

    Probes

    • Short single-stranded DNA sequences (around 30 nucleotides) used in hybridization.
    • Probes can be labeled for detection.
    • They are designed to specifically bind to complementary DNA sequences.

    Primers

    • Short oligonucleotide sequences (16-24 nucleotides) used as starting points for in vitro DNA synthesis.
    • They are required for DNA polymerase to synthesize new DNA strands.

    DNA Hybridization

    • Probes can bind to single-stranded DNA sequences.
    • Excess probe facilitates the hybridization process.
    • Hybridization is widely used for DNA detection and analysis.

    Polymerase Chain Reaction (PCR)

    • PCR is a technique used to amplify specific DNA sequences.
    • It involves repeated cycles of denaturation, annealing of primers, and extension by DNA polymerase.
    • Each cycle doubles the number of DNA copies.

    PCR Applications

    • Cloning of specific DNA or RNA fragments.
    • Detection of infectious agents using specific primers.
    • Analysis of short tandem repeats (STRs) for DNA fingerprinting.

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    Description

    This quiz explores the role of restriction enzymes in molecular biology, specifically their ability to cleave DNA into fragments and create sticky ends. It also covers techniques for DNA fragment separation using various gel electrophoresis methods and the application of plasmid vectors in recombinant DNA technology. Test your knowledge on these fundamental concepts of genetic manipulation.

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