Molecular Diagnosis 1+2 Flashcards
DNA gel electrophoresis
DNA travels through gel towards positive electrode (DNA is negatively charged) if placed in an electric charge
- largest size DNA does not travel far stays near well
- smallest size DNA can move further
Gel - a matrix that allows separation of DNA fragments
Buffer (NaCl) - allows charge on the DNA samples across the gel
Power supply - generates change difference across the gel
Stain/ detection - intercalates with the DNA molecule to identify the presence of the separated DNA
Why use restriction analysis
- to investigate the size of DNA fragments (e.g. small deletions)
- to investigate mutations (e.g. Sickle cell disease)
- to investigate DNA variation (e.g. DNA fingerprinting)
- to clone DNA
Cutting and rejoining of DNA
- same restriction enzyme reforms the restriction site when DNA ligase joins the sticky ends
- different restriction enzymes give the same complimentary sequence but the restriction site is not reformed
Gene cloning
A gene of interest is inserted into a plasmid
- carry genes to replicate independently
Multiple cloning site - MCS - restriction enzymes that are only present once
Recombinant DNA
- also includes a specific antibiotic resistance gene to eliminate bacteria that have not taken up plasmid by growing the cells in the presence of the specific antibiotic
Gene cloning
- cutting and joint of DNA of interest
- using vectors to carry the DNA of interest
- introducing vector plus DNA into suitable host cells in which replication will take place
- identification and isolation of clone containing the DNA of interest
Cloning human genes
- to make full proteins (e.g. insulin)
- to find out what genes do (e.g. HTT)
- genetic screening (e.g. CF)
- gene therapy (e.g. CF)
Mammalian cells better as they have the reverse transcriptase modification needed to produce mature insulin from pre-insulin
Polymerase Chain Reaction
PCR
Amplification of target DNA - exponential
Heat to 95C - DNA denatures
Cool to RT - DNA renatures
Add primers
Amplified DNA (forward and reverse - defines the region to be copied)
Thermostable DNA polymerase (Taq)
The breaking temperature depends on GC:AT ratio - more GC then lower melting temperature b
- for DNA profiling and DNA typing
Protein gel electrophoresis
- proteins are charged molecules and will move towards the anode or the cathode if placed in an electric field
- proteins can be separated on the basis of size, shape or charge
- serum protein electrophoresis - darker staining = more protein
SDS-PAGE
- separation of proteins on the basis of size
Isoelectric focusing (IEF)
- proteins separate on the basis of charge
- proteins migrate until they reach a pH equal to their pI
2D-PAGE
IEF followed by SDS-PAGE
- allows the separation of complex mixtures of proteins by charge and size
- important for diagnosing disease states in different tissues
Protein identification
proteomics
- digest protein with trypsin
- perform mass spectrometry
- generate list of peptide sizes
- use database of predicted peptide sizes for known protein to identify protein
Antibodies
Polyclonal antibodies
- produced by many b lymphocytes - specific to 1 antigen
Monoclonal antibodies
- produced from 1 b lymphocytes - specific to 1 antigen
Western blotting
to detect expression of proteins in mixtures
- gel
- binding of primary antibody
- binding of enzyme- linked secondary antibody
- immunoblot
Enzyme-linked immunoabsorbent assay
ELISA
- radioimmunoassay works in the same way but uses radio labelled primary antibody
Can be used to measure the concentration of proteins in solution e.g. Hormones
Enzyme assays
- to measure the rate of an enzyme catalysed reaction under defined conditions
Continuous assays - spectrophotometer pyruvate - measure the absorption of light
- chemoluminescence - glow in the dark
Discontinuous assays - radioactivity
- chromatography
Measure of enzymes
1) metabolic disorders in tissues
2) diagnosis of disease - serum enzymes
CLINICALLY IMPORTANT SERUM ENZYMES
AST - liver disease
ALT - liver disease
amylase/lipase - pancreatitis
Gamma-glutamyl transferase damage - liver disease
Alkaline phosphatase disorders - marker for bone
After actute MI
- diagnosis of MI is the measurement of cardiac troponin I by ELISA - antibody based reactions to measure
Measurement of metabolite concentrations using enzymes
E.g. Measurement of glucose concentration with glucose oxidase
- test strips H202 converted to coloured dye
- glucose monitor - glucose oxidase used as biosensor
Restriction enzymes
are molecular scissors - can isolate parts of DNA
Bacteria produce endonuclease
- recognition of degradation of foreign DNA
- specific endonuclease recognise and cut specific DNA sequences
- mostly palindromes (sequences that are the same from L-R and R-L)
- most
- produces staggered cuts, leaving sticky ends which can H-bond to each other
- protection of own DNA by methylation
