DNA Engineering and Cloning Quiz

DNA Engineering and Cloning

• Lecture Objectives: Learn about restriction enzymes, cloning DNA fragments in plasmids, transforming recombinant plasmids into bacterial cells, and different bacterial and eukaryotic vectors.
• Text Section: 7.1.20.1.
• Additional Reading: Material available on Canvas.

Cloning DNA/Gene

• Clone: An identical copy of a single mother cell.
• DNA/Gene Cloning Process:
  • Separates a specific gene or DNA fragment from a larger DNA molecule (e.g., chromosome).
  • Inserts the specific fragment into a vector (plasmid or carrier DNA).
  • Introduces the "construct" into a host cell.
  • Replicates the DNA in the host cell.

Restriction Endonucleases (REs)

• RE Function: Recognize and cut DNA at specific nucleotide sequences.
• RE Types:
  • Type I: Cuts DNA at a random site far from the recognition site (hundreds of bases). Recognition site is asymmetrical, with 3-4 nucleotides in one section and 4-5 nucleotides in another.
  • Type II: Cuts DNA within the recognition site. Recognition site is palindromic and 4-8 nucleotides long. Commonly used in DNA work.
  • Type III: Cuts DNA about 20-30 base pairs after the recognition site. Recognizes two inversely oriented non-palindromic sequences.
• Sticky Ends: Produced by some REs and can base-pair with each other or with complementary sticky ends.
• Blunt Ends: Produced by other REs.
• Base Cutters: Most frequently used REs in molecular biology are 6 base cutters.

Frequency of Type II RE Cutting

• Cutting Frequency Calculation: Y = 4ⁿ, where Y = expected number of cuts and n = number of bases in the recognition site.
  • 6 bp cutter: Cuts once every 4⁶ (4,096) base pairs, on average.
  • 4 bp cutter: Cuts once every 4⁴ (256) base pairs, on average.

Cloning DNA/Gene Steps

• 1. Select a vector (Fig 7-4).
• 2. Select appropriate restriction enzymes (REs) for the target gene and vector.
• 3. Digest the vector and target gene with the appropriate REs.
• 4. Ligate the target gene to the vector.
• 5. Transform the recombinant DNA into bacteria.
• 6. Select transformed bacteria.
• 7. Ensure host cells carry the recombinant DNA.

Cloning Vectors

• Purpose: DNA "vehicles" capable of self-replication, used for cloning, maintaining, and delivering DNA to biological systems.
• Types: Modified versions of naturally occurring plasmids (small DNA molecules in bacteria) or small viral DNA.
• Key Features:
  • Origin of replication (ori)
  • Selectable marker (e.g., TetR or AmpR)
  • Screenable marker (e.g., GFP and LacZ)
  • Multiple Cloning Site (MCS)
  • Relatively small size

Vector Classes

• i) Housekeeping: pBR322, pUC18/19 etc.
• ii) Expression: pET and pGEX vectors.
• iii) Delivery (Transformation): pCAMBIA (for plant transformation).

pUC19 Vector

• Ideal for: Gene cloning and maintenance.

Selecting RE Sites on DNA

• a) Locating Existing Sites: Software is available online (e.g., http://molbiol-tools.ca/) to find RE sites in a DNA sequence.
• b) Creating New Sites: PCR can be used (strategy 2).

Digesting DNA with REs

• REs cut at specific sequences, creating either sticky or blunt ends.

Ligation

• DNA ligases join DNA ends (strand breaks or nicks).
  • The 5' terminus must be phosphorylated.
  • The 3' terminus must have a free OH group.

Concatemer Concerns

• If only one RE is used, the target DNA may form a concatemer. This can create issues.

Transforming DNA into a Host Organism

• Methods: Heat shock, electroporation, conjugation, biolistics (gene gun).

Identifying Transformed Host Cells

• Selectable Markers: Genes (e.g., antibiotic resistance) that allow growth of cells under specific conditions (transformation).
  • Common Antibiotics: Ampicillin, kanamycin, and tetracycline.
• Screenable Markers: To visually identify cells and easier to select transformed colonies.
  • Example: LacZ gene.

Creating Sticky Ends via PCR

• Designed PCR primers
• Amplify the target gene in two separate reactions
• Purify/clean PCR products
• Mix products equally
• Denature and re-nature to generate desired sticky ends

Test your knowledge on DNA engineering, including the processes of gene cloning and the use of restriction enzymes. This quiz covers key topics from the lecture, focusing on the mechanisms of cloning and the role of various vectors in biotechnology.