DNA strands can be separated under conditions which break Hbonds

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• DNA strands can be separated under conditions
  which break H-bonds
• double-stranded molecules can be reformed in
  vitro
• formation of duplexes dependent on sequence
  complementarity
• homologous probes are used to detect specific
  nucleic acids

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Nucleic Acid Blotting

• Southern Blot
• described by Dr. Southern (1975)
• digest and electrophorese DNA
• transfer to membrane
• detect with probe
• Northern Blot
• electrophorese RNA
• Dot/Slot Blot
• no electrophoresis

• Examples of Probes
• previously cloned genes
• synthetic oligonucleotides

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Northern and Southern Blots
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Factors Affecting Hybridization

• temperature
• ionic strength
• chaotropic agents
• probe length
• probe mismatch
• GC

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Stringency and Melting Temperature

• Melting Temperature (Tm)
• temperature at which strands separate
• can estimate Tm with formulas

Effective Tm 81.5 16.6logNa 0.41(GC) -
0.65(formamide) 1.4(mismatch)
Synthetic Oligonucleotides Tm 2(A T) 4(G
C)
high Tm - 15o moderate Tm - 25o low Tm - 35o

• Stringency
• refers to relative conditions of the
  hybridization as compared to Tm
• reflects the homology between probe and target

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Labeling DNA Probes

• random priming
• used for cloned DNA fragments
• T4 nucleotide kinase
• used primarily for synthetic oligonucleotides
• (nick translation)
• earlier method replaced by random priming
• quality control and reproducibility problems
• (terminal transferase)
• adds nucleotides to 3 end
• used primarily for generation of homopolymer tails

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Random Priming

• purify DNA fragment
• kits available
• use ?-32P-NTP or other modified nucleotide
• boil probe before hybridization

Klenow modified DNA polymerase I
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T4 Polynucleotide Kinase

• transfers ?-PO4 from ATP to 5-OH
• major uses
• end-labeling (dephosphor-ylate first if
  necessary)
• phosphorylate synthetic oligonucleotides
• Maxim-Gilbert sequencing

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Non-Radioactive DNA Probes

• Enzyme-Linked Probes
• biotin/streptavidin
• digoxigenin-UTP/antibody
• enzyme cross-linking

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RFLP
Restriction Fragment Length Polymorphisms

• loss or gain of restriction site produces
  different sized DNA fragments on Southern blot
• also sensitive to insertion and deletion events
• polymorphisms do not have to be associated with
  the genetic locus of the probe
• application depends on enzymes and probes

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Diagnosis of Genetic Diseases by RFLP
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Genetic Fingerprinting

• use of repetitive DNA as probe produces complex
  RFLP patterns
• distinguish species, strains, individuals, etc.
• can be used in epidemiology, diagnosis, taxonomy,
  forensics, etc.

Isolates of Tuberculosis
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RNA Applications

• Northern blot
• sizes of transcripts
• gene expression information
• one gene at a time
• DNA Microarrays
• analyze multiple genes at one time
• whole genomes
• In situ hybridization
• combine with microscopy to localize cells
  expressing gene

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DNA Microarry (Gene Chips)

• different DNA probes are fixed onto solid
  surface (glass) in array
• fluorescent labeled target cDNA (mRNA) incubated
  with chip

• analyze 1000s of genes simultaneously
• identification of specific sequences
  (transcripts)
• expression levels (mRNA abundance)

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In Situ Hybridization

• incubate tissues with probe to detect cells
  expressing gene of interest

• 35S-label autoradiography
• fluorescent label FISH