Flashcards in MGD S7+8 - Molecular Diagnoses Deck (28):
Describe the process of DNA sequencingl
Fluorescently labelled dideoxyNTP (ddNTPs)
are added to a DNA template strand to create a complimentary DNA strand
2. Depending on what ddNTP is used the strand will terminate at different places
3. This produces lots of new DNA fragments of different lengths/sizes that can be denatured with heat and separated by gel electrophoresis.
Each of the ddNTPs can then be run in a separate lane and the end result allows us to write the DNA sequence.
Why are Dideoxynucleotides useful for DNA sequencing?
Due to removal of the 3' -OH group the ddNTPs cannot polymerise and cause chain termination during DNA polymerisation.
Describe the action of restriction endonuclease enzymes.
Bacterial restriction endonucleases recognise specific DNA sequences called 'restriction sites' and cut the DNA at this point.
Cutting of the DNA leaves exposed bases at the ends of fragments called 'sticky ends' (can be rejoined by DNA ligase)
What is restriction analysis useful for?
In conjunction with electrophoresis can be used to:
Investigate size of DNA fragments
Investigate DNA variation
Describe the process of gene cloning.
Plasmid is cut with restriction enzymes
Gene of interest is added with DNA ligase
This creates what is known as a recombinant DNA molecule
Plasmid is reintroduced into bacterium - Transformation
Bacteria containing Recombinant DNA multiplies
How might a researcher select for bacteria that have taken up a plasmid he introduced to the colony?
Plasmid often contains an antibiotic gene so a researcher can select for bacteria which have taken up the plasmid by adding an antibiotic to the culture.
What is DNA gel electrophoresis used for?
Hint: keep it general
To separate different sized DNA fragments
How is DNA gel electrophoresis performed?
1. Solution of different DNA fragments placed in a well at the negative cathode end of the gel
2. A charge is applied and the negative DNA moves toward the positive anode
3. Larger fragments move slower than smaller fragments and so fragments are separated by size.
4. Fragments of known size then used as a reference
What does the process of DNA gel electrophoresis require? (apart from DNA, smartass)
What is the Polymerase Chain Reaction (PCR) used for?
To amplify a specific DNA fragment
Investigate single base mutations
Investigate small insertions or deletions
How is PCR used to amplify a specific region of DNA?
Requires a specific sequence of cooling and heating DNA as follows
1. Denaturation of DNA at 94-96 degrees
2. Identifies region to be copied with a pair of primers that bind to specific regions on each strand
Renaturation (Annealing to primers) at 50-65 degrees
3. DNA polymerase is then used to copy the region being amplified.
DNA synthesis at 75-80 degrees
What is DNA hybridisation used for?
Give an example for each use
Investigate Gene structure
Eg. Large deletions/duplications
Investigate Gene expansions, triplet repeats
Eg. Fragile X syndrome
Eg. DNA fingerprinting
How is DNA hybridisation/Southern blotting preformed?
1. Begin with unlabbelled DNA that has undergone gel electrophoresis
2. DNA is transferred from the gel to a sheet of Nylon
3. The DNA fragments are then hybridised with a labelled gene probe to show the DNA sequences being looked for/at
What are the three styles of Blotting and what are they used for?
Southern blotting - DNA hybridisation
Northern blotting - Study of RNA
Western blotting - Analysis of proteins
How is PCR used in allele specific testing?
Use primers specific for sequence either side of allele of interest to amplify the allele
How is restriction analysis used in allele specific testing?
Use restriction endonucleases with restriction sites around the allele of interest
Analysis size of fragments produced to look for deletions/insertions etc
If endonuclease enzymes cut a wild type but not the sample we know the sample's restriction site is mutated/missing
How can DNA hybridisation used in allele specific testing?
Use a DNA probe complementary to wild type or mutated type, see which binds.
What methods of gel electrophoresis for proteins are there?
Describe the process of SDS-PAGE
The detergent SDS (sodium dodecyl sulphate) denatures proteins and gives the protein a negative charge proportional to molecular weight.
Electrophoresis then takes place, larger proteins that are more negative will travel further toward the cathode.
Describe the process of Isoelectric focusing
Proteins applied to a gel that contains a pH gradient
Electric field is applied
Proteins will migrate until it reaches a pH that matches its pI (isoelectric point)
At this point it will have no overall charge and will stop migrating
Describe in overview the process of 2D-PAGE
Proteins are applied to gel and are subject to SDS-PAGE and Isoelectric focusing (one at a time)
Between processes the gel is turned 90 degrees to separate bands out in a 2D pattern
What is an enzyme assay measuring and why?
Activity of an enzyme
Elevated levels/activity of various enzymes in serum is used as an indicator of tissue damage
Why are enzyme assays useful clinically?
They give an indication of whether an enzyme is present at normal levels
Under what conditions are all enzyme assays performed?
Optimal ionic strength
Appropriate ions and cofactors present
What is measured in an enzyme assay?
Rate of production of product or rate of disappearance of substrate
Give some examples of clinically important Serum enzymes and what disease they're associated with.
Hint: Don't bother memorising them all, just a couple examples
You know, if you'd like.
Aspartate/Alanine transaminase - Liver disease
Creatine kinase + Lactate dehydrogenase - Myocardial Infarction
Amylase/Lipase - Pancreatitis
gamma-glutamyl transferase - Liver damage, Increased by alcohol
Alkaline phosphatases - Bone disorders
Acid phosphatases - Prostate cancer
Plasma cholinesterase - Decreased in liver disease, Inhibited by organophosphate poisoning
What is Western Blotting?
Following SDS-PAGE, proteins are transferred to a nitrocellulose membrane and can be identified by binding with labelled antibodies