MGD S8 - Molecular Diagnosis Flashcards
Outline the Sanger Dideoxy Chain termination method
1) Denature DNA using heat
2) Add same labelled primer to the denatured template
3) A specific Fluorescent/radioactive stained DDNTPs are added (with regular dNTPs) along with DNA polymerase to create a complimentary strand
4) Depending on which DDNTP (ddATP, ddGTP, ddCTP, ddTTP) is used, the new strand will stop at different places
5) Repeated for all 4 DDNTP
6) This will produce fragments of different lengths that can be denatured with heat and then separated using gel electrophoresis
7) Each of the different ddNTPs run in different lanes allowing us to write the DNA sequence
8) Compare sequence with a reference sequence to spot differences
Explain restriction analysis
- Restriction endonucleases used to recognise specific DNA sequences; “restriction sites” and then cut the DNA Restriction endonucleases are bacterial enzymes
This together with gel electrophoresis can be used to:
- Investigate mutations (sickle cell disease)
- Investigate DNA variation
- Investigate size of DNA fragments (deletions and additions)
- Gene cloning
Explain gene cloning
1) Plasmids are used from bacteria - Small circular DNA - Can transfer to other bacteria - Can contain antibiotic genes
2) The plasmid is cut using restriction enzymes, gene of interest is added to create ‘recombinant DNA molecule”
3) Transformation; this is then introduced into the bacterium. Bacteria containing recombinant DNA are allowed to multiply
4) Allows rapid production of the gene of interest Plasmids often also contain an antibacterial resistance gene, in order to positively select for bacteria that have taken up the plasmid.
Explain gel electrophoresis
Used to separate different sized DNA fragments
1) A solution of different fragments is placed in a well at the negative cathode
2) A charge exerted by the anode encourages the negatively charged DNA to move towards the positive anode
3) Larger fragments move slowest
4) Fragments of known size are used as a reference This requires: gel, buffer, power supply and stain
Explain PCR
The polymerase chain reaction amplifies DNA segments by repeated copying of the target DNA using a thermo-stable DNA-polymerase and a pair of primers that uniquely define the region to be copied
1) Denaturation at high temperature (94-96 degrees celsius)
2) Renaturation (annealing) at lower temperature (50-65 degrees celsius)
3) DNA synthesis at medium temperature (75-80 degrees)
- This is used to amplify a specific DNA fragment - Investigate a single base mutation
- Investigate small deletions or insertions
What DNA polymerase is used in PCR?
Thermostable Tag thermos aquaticus
Describe southern blotting/ hybridisation
The unlabelled DNA from gel electrophoresis can be marked by southern blotting
1) Nylon used to transfer the fragments from electrophoresis
2) This is then hybridised with a labeled gene probe t show the specific DNA fragments
3) Use of radioactive probes can mark specific complimentary DNA fragments
This technique is used to:
- Investigate gene structure i.e Large deletions/ duplications
- Investigate gene expansion, triplet repeats Eg fragile X syndrome
- Investigate variation Eg DNA fingerprinting
What does northern blotting used to investigate
RNA
What does southern blotting used to investigate
DNA
What does western blotting used to investigate
Proteins
Explain SDS-Page
A technique that allows proteins to be separated purely on the basis of their molecular weight
1) The detergent SDS (sodium dodecyl sulphate) denatures protein molecules breaking the 3D structure and gives the protein a negative charge proportionate to molecular weight
2) Electrophoresis - largest molecular weight protein move the furthest (more negatively charged from the SDS)
Explain isoelectric focusing
Proteins can be separated y their isoelectric points (Pi) Proteins are applied to a gel containing a pH gradient, a protein will migrate until it reaches a pH that matched its Pi At this point it will have no overall charge so will stop migrating. Proteins with identical Pi values can be separated using 2D-PAGE
Explain 2D-PAGE
This allows the separation of proteins that have identical pI values but have different weights (or vice versa) The gell is turned by 90° and run for a different property to separate bands out. This is important technique for proteomics
What is aspartate transaminase (AST) and alanine transaminase (ALT) in serum a marker for?
Liver damage/disease
What is creatine kinase (CK) and Lactate dehydrogenase (LDH) in serum a marker?
Myocardial infarction
What is amylase/lipase in serum a marker for?
Pancreatitis
What is ɣ-glutamly transferase in serum a marker for?
Liver damage/disease Increased by alcohol
What is alkaline phosphate in serum a marker for?
Bone disorders
what is acid phosphatase in serum a marker for?
Prostrate cancer
In what conditions are enzyme assays performed at?
Optimal pH, temperature and ionic strength
Explain Enzyme linked immunoabsorbant assays
The concentration of a protein can be detected by analysing binding of its corresponding antibody
1) The first (primary) antibody (specific to protein) is mobilised on a solid support (e.g. micro titre well)
2) The solution to be assayed is applied to the antibody-coated surface
3) A second (secondary) antibody conjugated with an enzyme binds to the antibody-antigen complex
4) Binding to the second antibody is measured by assaying for the enzyme’s activity
Why are enzyme assays used?
- Measures enzyme activity
- Used as an indicator of tissue damage as enzymes normally contained in the tissues of elevated concentration in the plasma is indicative of tissue damage caused by a disease
How can restriction analysis be used in allele specific tests?
Use one or more restriction enzymes with restriction enzymes around or within the allele If restriction enzyme cuts the wild type but not the sample then the restriction site is missing or mutated
What is FISH
Fluorescent in situ hybridisation Used to detect abnormalities in DNA sequences on chromosomes in tissues through the hybridisation of fluorescently labelled probes
In what process is cDNA produced?
Reverse transcriptase-polymerase chain reaction (RT-PCR)
