DNA Cloning Notes

DNA Cloning

Basics of Cloning

  • Cloning involves copying a piece of DNA, such as a gene, many times over.
  • This process involves:
    • Taking a gene from our DNA.
    • Inserting it into a plasmid (a small, circular piece of bacterial DNA).
    • Using bacteria to multiply the gene through gene replication.
    • Using the bacteria to produce proteins via gene expression.

DNA and Plasmids: Similarities and Differences

Similarities:
  • Both DNA and plasmids are double-stranded molecules.
  • Each strand consists of a sequence of four nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T).
  • The nucleotides form hydrogen bonds with bases on the opposing strand to create the double helix.
  • Complementary base pairing:
    • A always pairs with T via two hydrogen bonds.
    • C always pairs with G via three hydrogen bonds.
Differences:
  • Human DNA is organized into 46 linear chromosomes.
  • Plasmids are circular, resembling a molecular DNA necklace.

Steps in DNA Cloning

1. Digestion with Restriction Enzymes
  • The DNA containing the target gene is digested using restriction enzymes.
  • Restriction enzymes bind to specific nucleotide sequences called restriction sites.
  • Example: EcoR1, which binds to the sequence GAA TTC and cleaves the DNA between G and the first A.
  • EcoR1 is used to cleave both the DNA containing the target gene and the plasmid DNA.
2. Creating Sticky Ends
  • Restriction enzymes cut the DNA at the restriction sites, creating sticky ends.
  • The target gene now has bits of the restriction sites on its ends.
  • The plasmid also has a gap with sticky ends.
3. Ligation
  • The target gene with sticky ends is combined with the plasmid that has sticky ends.
  • DNA ligase, an enzyme, is added to join the DNA fragments together, like puzzle pieces.
  • This creates a new hybrid DNA called recombinant DNA.
4. Antibiotic Resistance Gene
  • An antibiotic resistance gene is also inserted into the plasmid along with the target gene.
  • This allows for selective growth of bacteria containing the recombinant plasmid.
5. Transformation
  • The plasmids are transferred into bacteria (e.g., E. coli).
  • A stressor, such as heat shock, is applied to make the E. coli membrane more permeable to the plasmids.
  • Not all bacteria incorporate the plasmids.
6. Selective Growth
  • The bacteria are grown on an agar plate containing nutrients and antibiotics.
  • Bacteria that have not incorporated the plasmid lack the antibiotic resistance gene and die.
  • Bacteria that have incorporated the plasmid survive and multiply due to the antibiotic resistance gene.
7. Multiplication and Protein Production
  • As the bacteria divide, they replicate their DNA and the plasmid.
  • The number of plasmids (and the target gene) doubles with each bacterial division.
  • The bacteria act as protein factories, transcribing the target gene along with their own and producing the desired protein.

Practical Application: CFTR Gene and Cystic Fibrosis

  • Example: Cloning the CFTR gene, which codes for the cystic fibrosis transmembrane conductance regulator (a chloride channel).
  • In individuals with cystic fibrosis, the CFTR gene is mutated, resulting in either too little or an abnormally shaped protein.
  • This leads to problems with the pancreas and lungs.
  • Introducing normal CFTR proteins into patients via a vector has shown to improve lung function in some studies.

Summary of DNA Cloning Steps

  1. Digestion: Use restriction enzymes to cleave DNA containing the target gene and plasmid DNA at specific restriction sites.
  2. Ligation: Combine the target gene and plasmid DNA with DNA ligase to form recombinant DNA.
  3. Transformation: Transfer the plasmids into E. coli bacteria.
  4. Selection: Selectively grow bacteria containing the recombinant plasmid using antibiotic resistance.
  5. Multiplication/Expression: Allow bacteria to multiply, producing multiple copies of the target gene or its protein product.