Techniques to Study Nucleic Acids

• What is a restriction analysis?

  Analysis of DNA sequence fragments cut by restriction nucleases and separated by MW with gel electrophoresis
• What are restriction nucleases?

  Enzymes that recognize 4, 6, or more bases for cleavage site selection
• What do cleavage sites of restriction nucleases exhibit?

  A 2-fold axis of symmetry (5’ → 3’)
• How do restriction nucleases work?

  Bind as dimers (via charged amino acid residues) and cut phosphodiester bonds
• What are the two cut types of restriction nucleases?

  Endonucleases – cleaved within a sequence, Exonucleases – cleave from the end of a DNA (RNA) molecule
• What types of restriction enzymes require ATP?

  Type I and II
• How do Type I restriction enzymes cleave?

  Randomly
• How do type II and III restriction enzymes cleave?

  DNA chains at selected sites
• What are the two types of cleavage?

  “Sticky” (staggered) ends or “blunt” ends
• What is the cut location for “sticky” (staggered) ends?

  Either side of axis of symmetry
• What is the cut location for “blunt” ends?

  At axis of symmetry
• What is the term for restriction nucleases that recognize the same site?

  Isoschizomers
• How did early synthetic DNA production work?

  1. Denature and anneal with mixture of hexa-nucleotides
  2. Add primers (need 3’-OH)
  3. Add DNA polymerase and labeled nucleotides
• What is solid phase synthesis?

  Synthesis in the 3’ → 5’ direction, uses nucleotides with complex structures (activated monomer) that ensure proper order of addition
• What are the 3 different groups used in solid phase synthesis?

  DMT, βCE, and 3’-phosphoramidite
• What is the purpose of DMT in solid phase synthesis?

  Protects the 5’-OH
• What is the purpose of βCE in solid phase synthesis?

  Ensures correct phosphodiester bond is added
• What is the purpose of 3’-phosphoramidite in solid phase synthesis?

  Reaction leaving group
• What is the process (mechanism) of solid phase synthesis?

  1. Last base in sequence (growing chain) attached to resin (3’-end)
  2. Coupling of free 5’-OH (nucleophilic attack) and the activated monomer forming a phosphite triester intermediate
  3. Oxidation by I2 (carbonyl formation) to form phosphotriester intermediate
  4. Deprotection (removal of DMT) with dichloroacetic acid
  5. Repeat (until all nucleotides added)
  6. Remove βCEs and base protection
• What is southern blotting?

  Technique to identify (visualize) specific DNA sequences
• How does southern blotting work?

  1. DNA cleaved with restriction enzymes
  2. Gel electrophoresis run (size fractionation)
  3. Transfer DNA to nitrocellulose membrane (via capillary action)
  4. Hybridize with labeled DNA (P32) or RNA probe
  5. Expose to x-ray film
• What makes an RNA probe good?

  Only forms structures with specific gene/DNA of interest
• What makes an RNA probe bad?

  Forms structures with additional genes/DNA other than one of interest via imperfect base-pairing
• What is one use of southern blotting?

  Identification of blood at crime scene, can compared blood evidence to that of suspect/victim to determine how likely they are the culprit
• What is FISH used for?

  To identify the presence/absence of genes in a genome
• How does FISH work?

  Fluorescently tagged probes are hybridized to chromosomes and visualized using a fluorescence microscope
• What are the steps involved in Maxam-Gilbert Sequencing?

  Radiolabel DNA sample for identification, utilize different cleaving reagents for each sample, treat each sample with piperidine, run samples on gel to identify sequence
• What bases does dimethyl sulfate (neutral) cleave in Maxam-Gilbert sequencing?

  G > A
• What bases does dimethyl sulfate (acidic) cleave in Maxam-Gilbert sequencing?

  A > G
• What bases does Hydrazine + 1.5 M NaCl cleave in Maxam-Gilbert sequencing?

  C
• What bases does hydrazine cleave in Maxam-Gilbert sequencing?

  C + T
• What is the purpose of piperidine in Maxam-Gilbert sequencing?

  Cleaves phosphodiester bonds where bases have been removed
• How do you read the sequence from Maxam-Gilbert sequencing?

  From bottom to top (of gel), direct reading of template strand
• What are the disadvantages of Maxim-Gilbert Sequencing?

  Compounds are very toxic, some compounds are highly combustible or carcinogenic
• How does Sanger (Chain-terminator) sequencing work?

  Takes advantage of DNA polymerase reaction, utilizes low concentration of dideoxynucleotides to stop polymerization
• What are the requirements for Sanger (Chain-terminator) sequencing?

  DNA template, DNA polymerase, primer, dNTPs (excess), ddNTPs
• What is the mechanism (process) of Sanger (Chain-terminator) sequencing?

  1. Add ingredients to 4 different test tubes, each with a different base
  2. Generate different size fragments (due to random incorporation of ddNTPs)
  3. Run fragments on a gel
• How do you read the sequence in Sanger (Chain-terminator) sequencing?

  1. Read sequence is complementary to the template strand,
  2. Convert to corresponding bases (yields 3’-5’ template strand)
  3. Reverse order to get 5’-3’ template strand
• How does automated DNA sequencing work?

  Uses base terminators with fluorescent tags, different size fragment run through capillary electrophoresis
• What law does DNA sequencing efficiency follow?

  Moore’s Law → States that computer efficiency doubles every 2 years