Molecular Techniques Flashcards
What are restriction enzymes?
Endonucleases produced by bacteria
What are the functions of restriction enzymes?
Recognition and degradation of foreign DNA
Recognise and cut specific DNA sequences (restriction sites)
How do restriction enzymes cut DNA?
Break phosphodiester bonds between adjacent nucleotides - giving sticky ends
What is common in the sequences that restriction enzymes usually cut?
They usually cut palindromic sequences
What technique is used to separate DNA fragments created by restriction enzymes?
DNA gel electrophoresis
On what basis does DNA gel electrophoresis separate DNA fragments?
On the basis of size
What is required for DNA gel electrophoresis?
Agarose Gel - matrix that allows the separation of fragments
Buffer - allows charge on the DNA samples
Power Supply - generates charge difference across gel
Stain/Detection - to identify presence of separated DNA (UV)
Where do the DNA fragments in gel electrophoresis start? Why?
At the cathode (-ve electrode)
DNA is negatively charged due to phosphate groups moves towards the positive electrode (anode)
How are DNA fragments separated in DNA gel electrophoresis?
Larger fragments move less far in the gel
Smaller fragments move further in the gel
What can restriction analysis be used to investigate?
To investigate small deletions (size of DNA fragment)
To investigate mutations
To investigate DNA variation
To clone DNA
What vector is used in gene cloning?
Plasmid
What are some features of plasmids?
Small circular DNA found in bacteria
Can transfer to other bacteria (e.g. Antibiotic resistance genes)
What are the steps involved in gene cloning?
Isolate the DNA you want using restriction enzymes
Place DNA into plasmid using DNA ligase (producing recombinant DNA)
Introduce into suitable host cell (e.g. E. coli)
Identify and isolate the clone that contains the certain gene
What is a common application for gene cloning?
Making proteins (e.g. Insulin)
Are copies of genes initially taken in the RNA or DNA form? Why?
RNA form - doesn’t contain introns —> direct code for protein
Reverse transcriptase —> RNA —> DNA —> plasmid
What is PCR useful for?
Amplification of a target DNA sequence
What three things does PCR require?
A thermostable DNA polymerase (Taq polymerase)
Specific forward and reverse primers
Temperature cycling - denature, anneal, polymerise
What is PCR useful for investigating?
Single base mutations
Small deletions
Genetic variation
What are the 3 steps involved in PCR and their temperatures?
Denaturing (95 degrees Celsius)
Annealing (55 degrees Celsius)
Polymerisation (72 degrees Celsius)
What is the function of protein gel electrophoresis?
To separate proteins based on size, charge and shape (usually size)
What 4 things are required for protein gel electrophoresis?
Gel
Buffer
Power supply
Stain
How does protein gel electrophoresis work?
Contents of cell (mixture of macromolecules) placed at wells near cathode in a vertical gel
Larger proteins move less far in the gel
Smaller proteins move further in the gel
When is SDS-PAGE used?
When you want to ignore the intrinsic properties of a protein and separate by size only
How does SDS-PAGE work?
Protein unravelled —> using detergent; SDS and beta-ME
Then separated on a gel using protein gel electrophoresis
What is isoelectric focusing used for?
To separate proteins based on their isoelectric point (charge)
What is the isoelectric point of a protein?
The pH at which the protein carries no charge
How is isoelectric focusing carried out?
Ph gradient created in a gel with an electric field
Proteins migrate till they reach the pH that equals their pI
What is 2D-PAGE used for?
Used to separate complex mixtures of proteins based on charge and size
Important in diagnosing disease states