Molecular Techniques Flashcards
How do restriction enzymes work?
specific endonucleases recognise and cut palindromic recognition sequences
How does gel electrophoresis work?
gel runs from negative electrode to positive, dna is negatively charged so it runs up the gel
smaller fragments of DNA move further than larger fragments
restriction endonucleases cut dna into fragments of varying size
When is restriction analysis appropriate?
to investigate the size of DNA fragments (e.g. small deletions)
to investigate mutations (sickle cell disease)
to investigate dna variation (dna fingerprinting)
to clone DNA
How does gene cloning work?
gene of interest isolated using restriction enzyme
gene of interest inserted into plasmid vector (recombinant DNA)
recombinant DNA inserted into suitable host cell
identify and isolate the clone containing the DNA of interest
How is gene cloning useful?
finding out what genes do
genetic screening e.g. Huntington’s, Cystic Fibrosis
gene therapy
What things does PCR require?
heat stable taq polymerase
pair of primers (forward and backward), uniquely defining region to be copied
How does PCR work?
1) Denaturation (95c)
2) Primers added
3) Primer extension (72c/Taq Polymerase)
When is PCR useful?
investigating single base mutations
investigating small deletions or insertions
investigating variation, genetic relationships e.g DNA profiling
How does protein gel electrophoresis work?
proteins are charged molecules and will move towards the anode or cathode if placed in an electric fluid
proteins can be separated on the basis of size, shape or charge
How does SDS page work? (form of protein gel electrophoresis)
separation of proteins on the basis of size
detergent breaks disulphide binds so you examine the primary structure only
How does isoelectric focusing work? (form of protein gel electrophoresis)
proteins separate on the basis of charge by running through a gel which separates on basis of pH
How does 2D-PAGE work?
- allows separation of complex mix of proteins
- e.g pH and size at the same time
What is proteomics?
digesting protein with trypsin and performing mass spectrometry to work out size. then, comparing against a database of peptide sizes.
What is Western Blotting?
Artificial antibodies are created that react with a specific target protein. The sample to be tested is prepared and put together with these antibodies on a membrane – if the specific protein sought for is present, after a gel electrophoresis step this will result in an accordingly stained band on the western blot
How do enzyme linked immunoabsorbant assays work?
Can be used to measure the concentration of proteins in solution e.g. hormones
- antigens in well
- specific antibodies added
- enzyme linked antibodies added which binds to primary antibodies
- substare added, rate of colour formation equivalent to amount of specific antibody
What do enzyme assays do?
measure the amount of product
What are some clinically important markers of disease (enzymes) in the body?
Alanine transaminase (ALT)/Aspartate transaminase (AST)-Markers for liver damage / disease
Amylase/ lipase- Marker for pancreatitis
g-glutamyl transferase- Marker for liver damage Increased by alcohol
Alkaline phosphatase- Marker for bone disorders
How does DNA hybridisation work?
Denaturing DNA
adding fluorescent probe
and reannealing
How does southern blotting work?
digest dna with restriction enzymes
separate dna fragments by gel electrophoresis
transfer single stranded dna fragments to nylon
hybridise filter with labelled gene probe
detect hybridisation (and hence dna of interest) via exposure filter to x-ray film
When is southern blotting useful?
Southern blotting allows us detect pieces of DNA from complex mixtures that would otherwise be very difficult to detect.
good for large deletions or duplications
It also allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.
In many cases we can use Southern blotting in combination with PCR to detect things like gene structure, gene expansions and repeats, mutations and genetic variation etc. (allele specific probes)
How does DNA sequencing work?
by adding dideoxy nucleotides to DNA fragments, the chain terminates at different places. DNA fragments of different sizes produces. By using a gel electrophoresis, a sequence can be read from top to bottom.
How does RT-PCR work?
gene expression measured (mRNA examined)
RNA template converted into DNA and then amplified
How does microarray technology work?
analysis on a genome wide level, checks gene expression (red vs green)
good for comparison, works via hybridisation principles
How does array competitive genome hybridisation work?
checks for micro deletions and duplications by comparisson of sample DNA to normal dna
How does DNA fingerprinting work?
examines non coding areas of the genome, by examine micro satellites (repeated areas of 30-60 bases)
Restriction enzymes and southern blotting with probes for micro satellites can show genetic links.
What is FISH?
denatured DNA labelled with fluorescent dye and hybridise. It highlights regions within DNA via labelled probes (chromosome painting, can show translocations)
