1. 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

2. Nucleic Acid Blotting Southern Blot Northern Blot Dot/Slot 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

3. Northern and Southern Blots

4. Transfer DNA/RNA to Membrane
Capillary Action
original method
no special apparatus
efficient, but slow
Vacuum
special apparatus
quick and efficient
Electrophoretic
not widely used
Fix Membrane
‘baking’ (heat 80o)
UV cross-link

5. Northern and Southern Blots

6. Factors Affecting Hybridization
temperature
ionic strength
chaotropic agents
probe length
probe mismatch
% GC

7. Stringency and Melting Temperature
Melting Temperature (Tm)
temperature at which strands separate
can estimate Tm with formulas:
Effective Tm = log[Na+] (%GC) (%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

8. Labeling DNA Probes random priming T4 nucleotide kinase
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

9. Random Priming purify DNA fragment kits available
use -32P-NTP or other modified nucleotide
boil probe before hybridization
Klenow = modified DNA polymerase I

10. 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

11. Non-Radioactive DNA Probes
Enzyme-Linked Probes
biotin/streptavidin
digoxigenin-UTP/antibody
enzyme cross-linking

12. 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

13. Diagnosis of Genetic Diseases by RFLP

14. Genetic ‘Fingerprinting’
use of repetitive DNA as probe produces complex RFLP patterns
distinguish species, strains, individuals, etc.
can be used in diagnosis, taxonomy, forensics, etc.
Isolates of Tuberculosis

15. 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

16. DNA Microarry (Gene Chips)
different DNA ‘probes’ are fixed onto solid surface (glass) in array
fluorescent labeled target cDNA (mRNA) incubated with chip
analyze 1000’s of genes simultaneously
identification of specific sequences (transcripts)
expression levels (mRNA abundance)

17. In Situ Hybridization
incubate tissues with probe to detect cells expressing gene of interest
35S-label + autoradiography
fluorescent label = FISH