DNA Technology

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DNA Technology

• Chapter 21

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DNA Sequencing

• DNA replication occurs by adding nucleotides to
  the 3 hydroxyl group
• Dideoxynucleotide triphosphate terminates
  reaction
• Radioactive 32P used for detection
• Fluorescence probe used in place of the 32P
• Separation based on size (gel electrophoresis)
  one base pair difference between fragments

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Fluorescence Methods

• Single fluorescent primer and nonfluorescent
  ddNTPs four separate lanes (each terminated
  with respective ddNTP)
• Different fluorescent probe on each ddNTP run
  in same lane and identify by emission spectra of
  the base
• Need distinct emission spectra and excitation at
  same wavelength

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ddNTP Terminators

• Can be excited at 488 QY differ by less than a
  factor of 2.
• Emission spectra overlap (need to use two
  different band pass filters to identify bases)
• Capillary electrophoresis identify 1000
  bases/hour
• Alternative methods FRET or Lifetimes

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DNA Hybridization

• Based on FRET between donor and acceptor labeled
  DNAComplementary strands
• Binding to adjacent regions along same DNA strand
• Donor intercalates with double stranded DNA and
  interacts with acceptor labeled oligo
• Competitive hybridization

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FISH

• Fluorescence in situ hybridization metaphase
  chromosomes are exposed to probe DNA
• Denaturation of chromosomes and hybridization
  with probe DNA
• Fluorophores should not interfere with
  hybridization
• Fluorophores can be incorporated into DNA without
  disrupting base pairing
• Ability to detect all 24 chromosomes
• Sort by flow cytometry
• Identify specific sequences associated with
  disease or markers