Biotechnology Quiz: Recombinant DNA Technology

Recombinant DNA Technology

• Involves selecting a desired gene and a suitable vector for integration into a host.
• DNA ligase joins DNA fragments with cloning vectors.
• Restriction endonucleases recognize specific sequences (restriction sites) to cut DNA.
• Endonucleases are isolated from various microorganisms.
• Terminal transferase converts blunt ends of DNA fragments into sticky ends.
• DNA polymerase synthesizes complementary nucleotides.
• Alkaline phosphatase removes terminal phosphate groups.
• Polynucleotide kinase adds phosphate groups to terminal 5' ends.
• Recombinant DNA technology has five basic steps: culturing host cells, extracting products, downstream processing of the products.
• Gene splicing involves cutting DNA at a specific site.
• Vectors carry foreign genes into host cells.
• Transformation is the introduction of free DNA into bacteria.
• Competent cells can take up DNA.
• Electroporation uses an electric shock to disrupt cell walls in order to allow DNA uptake.
• Transformed cells have new DNA.
• Marker genes identify successfully transformed organisms.
• pBR322 is a plasmid vector.
• tet gene confers resistance to tetracycline.
• amp gene confers resistance to ampicillin.

Applications of Recombinant DNA Technology

• Recombinant proteins are used in laboratory experiments and to generate antibodies.
• Bacteria can produce human insulin and growth hormone.
• Bacteria can be engineered to consume oil spills.
• Recombinant DNA technology has applications in agriculture.

DNA Extraction

• DNA extraction can be done from nucleated cells.
• The goal of DNA extraction obtains a relatively pure form for further analysis.
• DNA extraction procedures have two main parts: lysing the cell and removing contaminants.
• The technique involves: lysis, precipitation, wash, re-suspension
• Detergents are used to disrupt the plasma membrane.
• Proteases and RNAases are used to eliminate proteins and RNA.
• Supernatant (containing DNA) is transferred to a clean tube and is precipitated using ethanol.
• Methods include the use of detergent/enzymes, centrifugation, and precipitation.

Gel Electrophoresis

• Separates molecules based on charge and size in a gel.
• Charged molecules migrate in an electrical field through a gel matrix.
• Gel's porosity affects migration speed.
• Molecules migrate towards the opposite charge.
• Used for separating DNA fragments and other biological molecules.
• Gel electrophoresis uses buffers (e.g., TAE, TBE) to maintain constant pH and support current flow.
• DNA is negatively charged.
• Smaller pieces travel further than larger ones.

PCR (Polymerase Chain Reaction)

• PCR amplifies specific DNA sequences.
• PCR uses a thermostable DNA polymerase (e.g., Taq polymerase) and specific primers.
• PCR steps include denaturation, annealing, and extension.
• Denaturation creates single-stranded DNA.
• Primers anneal to the template DNA.
• Taq polymerase extends the primers along the template DNA.

Blotting Techniques

• Transferring DNA, RNA, or proteins from a gel to a membrane for analysis.
• Southern blotting detects DNA.
• Northern blotting detects RNA.
• Western blotting detects proteins.
• Techniques are helpful to study mutations in genes
• Advantages: good sensitivity, multiple protein analysis, and detection of mutations.