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DNA strands can be separated under conditions which break H-bonds

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Presentation on theme: "DNA strands can be separated under conditions which break H-bonds"— Presentation transcript:

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

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