Molecular Techniques. Studies of cell Fractionation Purification/ Identification Structure/ Function. Proteins. Carbohydrates. Lipids. Nucleic acids. Organelle level. Cell fractionation Nucleus Mitochondria RER, cell membrane SER Cytosol. Cellular level. Microscope.
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Molecular level: Macromolecules
C, H, O, N, S, P
A centrifuge working at speeds in excess of 20,000 RPM is an “ultracentrifuge”.
Centrifugation force to pellet the cellular components
Molecules separate according to size and shape
What make rate-zonal and isopycnic centrifugations difference?
+ - - -
Agarose gel staining
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2-dimensional Gel Electrophoresis
Detect the expression level and transcript size of a specific gene in a specific tissue or at a specific time. Sometimes mutations do not affect coding regions but transcriptional regulatory sequences (e.g., UAS/URS, promoter, splice sites, copy number, transcript stability, etc.)
Components of the gel are then transferred to a solid support or transfer membrane
Wet filter paper
Add antibody against yours with a marker (becomes the antigen)
Stain the bound antibody for colour development
It should look like the gel you started with if a positive reaction occurred
Rinse with ddH2O
Add monoclonal antibodies
Antibodies will bind to specified protein
Background on PCR
Double Stranded DNA is denatured by heat into single strands.
DNA polymerase catalyzes the production of complementary new strands.
The process is repeated for each new strand created
1. Usually fewer than 3000 bp
2. Identified by a specific pair of DNA primers- usually oligonucleotides that are about 20 nucleotides
Magnesium as a Cofactor
Stabilizes the reaction between:
Denatures DNA by uncoiling the Double Helix strands.
How PCR works
Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.
Next, denature the DNA at 94˚C.
Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA.
Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus).
Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA.
Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.
Cool to 4˚C and store or use amplified PCR product for analysis.
Thermal cycler protocol Example
Step 1 7 min at 94˚CInitial Denature
Step 2 45 cycles of:
20 sec at 94˚CDenature
20 sec at 64˚CAnneal
1 min at 72˚CExtension
Step 3 7 min at 72˚CFinal Extension
Step 4 Infinite hold at 4˚CStorage
The Polymerase Chain Reaction
THE REACTION COMPONENTS
Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
Use specific programs
PRIMERDESIGNERSci. Ed software
Also available on the internet
1 1.5 2 2.5 3 3.5 4 mM
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides
USE MASTERMIXES WHERE POSSIBLE
1. Gene isolation
2. Fingerprint development
Applications of PCR
Sequencing of DNA by the Sanger method