Презентация на тему: PCR and sequence
Hybridization (DNA-DNA or DNA-RNA)
HYBRIDIZATION? – Yes, it is about this familiar picture
We can denaturate and renaturate DNA by heating/cooling
As DNA is heated, it reaches a temperature where the strands separate (DNA melts).
Tm of DNA is affected by:
RNA can bind DNA (U is equivalent of T in hybridization)
Hybridization could be less than perfect
So, hybridization is a most obvious phenomenon to use for specific DNA detection
Polymerase labeling of DNA
T4 polynucleotide kinase labeling
Professor Sir Ed Southern, Whitley Professor of Biochemistry at the University of Oxford
“Real” Southern blot (DNA-DNA blot)
Colony hybridization assay for the identification of bacterial colonies carrying a particular DNA clone
Design of degenerated synthetic hybridization probes (for already known proteins only)
The degeneracy of a primer is the number of unique sequences it corresponds to (5 in one of the examples below).
Fluorescent probe hybridization
How streptavidin/biotin binding is working?
IN SITU HYBRIDIZATION is an imaging method to visualize mRNA expression in tissues and cells.
Cy3/Cy5 direct labelling of DNA (for microarrays)
2. Polymerase chain reaction (PCR)
Polymerase Chain Reaction (PCR)
Exponential nature of PCR amplification
Avoid primer dimer formation
Use Software to avoid of such problems
Typical PCR gel (Every PCR should by gel-verifyed)
Non-specific PCR and how to improve it
PCR enzymes
Tth polymerase
Pfu polimerase
Pol Vent (From Thermococcus litoralis)
Long-Range PCR
3. SEQUENCING: (Sanger method)
Dideoxynucleotide blocks chain elongation
DNA sequencing: chemistry, with terminator dyes
Semi-automated fluorescent DNA sequencing:
Applied Biosystems Inc., have designed an automated method that combines the PCR and actual sequencing
DNA sequencing by primer walking
Chemical synthesis of DNA
General consideration about Gene Expression Expression Host -> Expression System Promoter system -> expression vector Properties of product -> stability Production level
Comparison of expression systems
Prokaryotic Expression vector
Prokaryotic Expression vector
Eukaryotic Expression vector
Promoters
Control of transcription of the lac operon.
