Molecular Biology Techniques Flashcards
What are restriction enzymes
Endonuclease cleaves DNA on recognizing a specific sequence (4-6 base sequences long)
What is gel electrophoresis
Fragments of DNA can be separated
Loaded into well in tray of agarose gel across which an electric field is applied
DNA negatively charged and will move towards the positive charge
Small fragments migrating more quickly than large fragments
UV light visualises DNA after staining with ethidum bromide
What is PCR?
Amplify short segments of DNA
DNA polymerase repeatedly replicates a sequence of DNA
Taq polymerase synethsises DNA in 5’ to 3’ direction
REquires primer to initiate the synthesis
Deoxynulceotide triphophate substrate used by Tap polymerase to synethsise new strands of DNA
5’ and 3’ primers used to start the process of replication by binding to complementary sequnces on opposing DNA strands
Buffer maintains optimum pH
Process of PCR
Reagents combined
Heating and cooling cycles
(can be done from RNA but requires reverse transcriptase to generate DNA strand for PCR reaction
How can a sequence of DNA be identified?
Nucleic acid hybridisation
Probe sequence labelled and added to mixture with target sequence
Both sequences denatured to single strand form
Probe sequence binds to complementary sequence
Hybrids detected by autoradiography (if radioactively labelled) or be chromogenic detection(with fluorescent probes)
What is southern blotting?
Detection of specific DNA sequence
Transfer of electrophoresis separarted DNA fragments on agarose gel to a filter membrane and subsequent fragment detection by probe hybridisation
What is Northern blotting
Study expression of genes
mRNA are separated by electrophoresis and transferred to a nylon or nitrocellular membrane - used for hybridisation experiments
What is Western blotting?
Gel electrophoresis used to separate denatured proteins
Transferred to nitrocellulose membrnae for using fluorescently labelled antibody which binds target protein
