Polymerase Chain Reaction Overview

Polymerase Chain Reaction

• PCR amplifies specific DNA sequences (up to 6 kb)
• PCR requires:
  • Heat-stable DNA polymerase (e.g., Taq polymerase) for nucleotide addition (5'→3')
  • Deoxynucleoside triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP
  • Two oligonucleotide primers flanking the DNA segment of interest
• Denaturation: Separating dsDNA into single strands using heat to break hydrogen bonds
• DNA polymerase requires a free 3'-OH group on the template strand for nucleotide addition.
• Primers bind to the 3' end of the template strand, allowing DNA polymerase to add nucleotides.
• Amplified strand is complementary to the template strand and synthesized in the 5'→3' direction.
• Each PCR cycle doubles the amount of target DNA.
• At the end of 1 cycle, there are 2² strands of DNA.
• Number of DNA molecules after 'n' cycles = 2^(n+1)
• PCR applications:
  • Sequencing (e.g., forensics, infectious disease diagnosis, mutation detection)
  • Cloning (e.g., studying models, therapeutics)

Recombinant DNA Technology

• Isolates, amplifies, and modifies specific DNA sequences.
• Also known as molecular cloning or genetic engineering.
• Cloning involves producing multiple identical organisms from a single ancestor.
• A clone is a collection of cells containing a vector carrying the DNA of interest.
• Cloning aims to:
  • Produce large amounts of DNA
  • Express large amounts of protein

Overview of Cloning

• Steps:
  • Generate DNA fragment of interest using restriction enzymes, PCR, or chemical synthesis.
  • Ligation: Insert fragment into a vector (another DNA molecule) containing sequences for replication.
  • Transformation: Introduce the vector (with DNA of interest) into host cells for replication.
  • Selection/screening: Identify and select cells with the desired DNA.

Cloning: Vectors

• Small, autonomously replicating DNA molecules.
• Plasmids are circular DNA molecules (up to 200 kb) found in bacteria or yeast.
  • Present in hundreds of copies within the cell
  • Small and replicate easily
  • Carry genes encoding antibiotic resistance for selection.
  • Carry restriction endonuclease sites for foreign DNA insertion.
  • Can clone up to 10 kb.
  • Example: pUC18

Cloning: Ligation

• A restriction fragment is inserted into a cut made in a cloning vector by the same restriction enzyme (sticky ends).
• Complementary ends of the DNA fragment and vector base pair (anneal).
• The sugar-phosphate backbone is covalently joined by DNA ligase.
• The inserted fragment of foreign DNA can be excised using the same restriction enzyme.

Cloning: Transformation

• A chimeric plasmid is taken up by a host bacterium (transformation).
• The plasmid becomes permanently established and replicates indefinitely in the host cell.
• This produces large amounts of recombinant DNA.
• Bacteria are typically cultured on semi-solid growth medium at low density to enable the formation of single colonies.
• Assumption: One colony arises from a single cell.
• Only host cells containing a properly constructed vector are selected for further propagation.

Cloning: Selection

• Methods for selecting positive transformants:
  • Antibiotic resistance: Use antibiotic genes (ampR, tetR, kanR)
    • Untransformed cells do not grow on antibiotic media; transformed cells do.
  • Chromogenic substances:
    • Nucleic acid gel electrophoresis: Separates nucleic acids by size.
    • In an electric field, the velocity of a charged molecule is proportional to charge density, size, and shape.
    • Nucleic acids have constant shape and charge density; velocity depends on size.

Nucleic Acid Gel Electrophoresis

• Separates nucleic acids based on size using a gel-like matrix (agarose or polyacrylamide).
• Molecules are applied at one end of an electric field and migrate to the other end.
• Smaller molecules move through the pores faster and migrate farther.
• Visualized using stains:
  • Dyes binding to DNA
  • Radioactive labeling
  • Fluorescence
• A molecular weight ladder (set of standards of known sizes) can be added to estimate size.
  • Derived from restriction enzyme cleavage of a known DNA sequence, PCR, or DNA ligation.

DNA Fingerprinting

• Used to identify individuals based on their unique DNA profiles.
• Relies on differences in the length of restriction fragment length polymorphisms (RFLPs).
• RFLPs are created by cutting DNA with restriction enzymes.
• Different individuals have different RFLP patterns due to variations in their DNA sequences.
• Can be used for:
  • Paternity testing
  • Forensic investigations

Restriction Enzymes

• Enzymes that cut DNA at specific recognition sequences.
• Each restriction enzyme recognizes a unique sequence of nucleotides.
• Examples:
  • EcoRI: G↓AATTC
  • HaeIII: GG↓CC
  • BamHI: G↓GATCC
  • PvuII: CAG↓CTG