Introduction To Molecular Techniques Flashcards
Role of restriction enzymes
- they recognise speechifying sequences of DNA known as palindromic sequences.
- they cut DNA at these specific sites
What is a palindromic sequence
- a sequence that is the same when read from the 3’ to 5’ and 5’ to 3’ direction
What are the 4 requirements for gel electrophoresis
1) gel : ( often agorose gel) which is a matrix that allows separation of DNA fragments
2. Buffer : allows maintenance of the charge on the DNA samples across the gel
3. Power supply : generates charge difference across gel
4. Stain/detection : to identify presence of separated DNA.
Where on the electrophoresis gel plate is DNA placed ?
At the negative electrode because it is negatively charged so would need to move towards the positive electrode
Why do we use gel electrophoresis?
1) to investigate the size of DNA fragments
2. To investigate mutations
3. To investigate DNA variations
4. To clone DNA
What are the5 basic steps of cloning a gene
1) isolate relevant gene of interest following digestion with restriction enzymes
2. Insert gene of interest into plasmid vector - same restriction enzyme is used to cut the vector.
3. Ligation : DNA ligase forms RECOMBINANT DNA by forming phosphodiester linkages between the nucleotides of vector and gene.
3. Introduce recombinant dna molecule into suitable host cells
4. Identify and isolate the clone containing the DNA of interest
Why do we clone human genes ?
1) to make useful proteins eg insulin
2. To find out what genes do
3. Genetic screening
4. gene therapy
Outline the steps in polymerase chain reaction
1) denaturalising: the strands must first be separated. DNA is heated to 95 degrees - this breaks the hydrogen bonds and causes DNA to unwind.
2. ANNEALING : temp is lowered to around 50 degrees to allows PCR primers to bind to the exposed strand of DNA.
3. Extension : the process of adding new complementary nucleotides using Taq polymerase ( a thermostable polymerase which can tolerate the high temperatures). The temperature is raised to 75 degrees. This forms duplicate double helix.
Wha what is the function of PCR?
- Amplification of target DNA
- investigate single base mutations
- to investigate small deletions or insertions
- investigate variations
On what basis are proteins separated?
- size
- shape
- charge
- they will move towards the anode or cathode if placed in an electric field
What are the requirements for protein gel electrophoresis
- Gel : a matrix that allows separation of the protein sample
- Buffer : maintains charge on the protein samples
- Power supply : generates a charge across the gel
- Stain / detection : to identify the presence of separated proteins
What is the role of serum protein electrophoresis?
- measures specific proteins in the blood to help identify some diseases.
- serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups of similar size , shape and charge.
Why SDS-PAGE separates proteins on the basis of size ?
SDS separates proteins primarily by mass because the detergent ( SDS) denatures and binds to proteins to make them uniformly negatively charged.
- thus when a carried is applied , all SDS bound proteins in a sample will migrate through the gel towards the positively charged electrode
Why do we use enzyme assays ?
- important tools for measuring cellular activity of enzyme kinetics.
- provides us useful information of the mechanism of enzyme catalysis and interactions of enzymes with substrates , drugs etc.
How will you measure the product in continuous enzyme assay ?
- chemoluminescence
- spectrophotometery
How would you measure the product during discontinuous enzyme assay ?
- radioactivity
- chromatography
How can antibodies be used in immunoassays to detect the presence of a protein?
- it is used to detect and quantify the amount of protein.
1. An antigen must be immobilised first to a solid surface,
2. Antibody that is linked to an enzyme then binds to the antigen.
3. Incubated with a substrate to produce a measurable product. Enzyme converts substrate into coloured product. The rate of colour formation is proportional to the amount of specific antibody
4. The more antibodies that bind = more protein there is.
How can antibodies be used in western blotting to detect the presence of proteins ?
- protein gel is transferred on a nylon membrane
- Add antibody which binds to specific protein
- Use another antibody ( eg fluroscent one) to detect first antibody .
Where do restriction enzymes come from ?
Bacteria
What type of primers do we need during the annealing process of PCR?
- we need primers that go in the opposite direction , not the same direction,
- they need to actually span the gene on the DNA strand
Outline the steps of southern blotting - a technique of DNA hybridisations
1) digest DNA with a restriction enzyme
2. Separate DNA with gel electrophoresis
3. The gel is soaked in an alkaline solution which denatures the DNA into ssDNA molecules ( single stranded).
4. The separated fragments of ssDNA now need to be transferred to a solid support : usually nylon or nitrocellulose filter is used.
5. Once the DNA has been transferred onto the nylon filter the gel can be discarded.
6. The filter is now placed in a solution containing labelled DNA probe ( Radioactive or fluorescent labelled). These probes will bind to ssDNA on the filter with a complementary sequence.
7. After binding the probe , we wash the filter to remove any unbound probe.
8. Detect binding using photographic film or UV light for fluorescent detection methods.
Why do we use southern blotting ?
- To investigate variation
- Investigate mutations
- Investigate gene expansions ( triplet repeats)
- Investigate gene structure ( large deletions or duplications)
- Allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.
What are important characteristic of DNA probes in southern blotting ?
- the probes do not have to have 100% similarity to the target sequence - it will just bind less tightly
2. Probes do not have to completely align with the sequence they are being used to detect
3. Probes do not affect the position of the target sequence on a gel
What is the Sanger chain termination method
- allows us to work out the nucleotide sequence of a piece of DNA
