Restriction Enzymes, Cloning Vectors & DNA Profiling – Review

Restriction Enzymes & Sticky Ends

• Restriction enzymes cut at specific recognition sequences, leaving “sticky ends” (single-stranded overhangs).
• Overhang direction defines type:
  • 5' overhang – protruding strand points toward the 5' end.
  • 3' overhang – protruding strand points toward the 3' end.

Determining Overhang Type

• Locate the single-stranded extension.
• Trace its direction along the backbone; the end it approaches ( 5' or 3' ) names the overhang.

Fragment Prediction & Counting

• Circular DNA: fragments = number of cut sites.
• Linear DNA: fragments = cut sites +1.
• Mixed digests (two or more enzymes) → fragments correspond to all unique cut positions.
• EcoRI example: produces a 5' overhang and two fragments at its single site.

Common Cloning Vectors

• Plasmids
• Bacteriophages
• Cosmids
• BAC (Bacterial Artificial Chromosome)
• YAC (Yeast Artificial Chromosome)

Essential Plasmid Features

• Origin of replication (ori)
• Antibiotic-resistance gene(s)
• Selectable marker (e.g. lacZ for blue/white screening)
• Multiple restriction sites (MCS)
• Total plasmid size (kbp) stated on map

Plasmid Mapping Basics

• Start with single digests to identify number/size of fragments ⇒ locate individual sites.
• Double digest combines sites; overlapping fragment sizes position sites relative to each other.
• Single site → one linear fragment of full plasmid size.
• n sites in circular plasmid → n fragments; sizes must sum to total plasmid length.

DNA Profiling Techniques

• RFLP (Restriction Fragment Length Polymorphism)
  • Uses restriction enzyme digestion + gel electrophoresis.
  • Requires large DNA quantity; slower.
• VNTR (Variable Number Tandem Repeat)
  • Similar to RFLP but targets tandem-repeat regions; moderate DNA needed.
• STR (Short Tandem Repeat)
  • PCR amplifies repeat loci with fluorescent primers; minimal DNA; rapid.
  • Capillary electrophoresis for sizing.