Molecular Diagnosis Flashcards
What can we use to analyse proteins?
protein electrophoresis, immunoassays and enzyme assays
Why do bacteria naturally produce endonucleases?
It is the normal mechanism for recognising and degrading foreign DNA in bacterial cells.
How do bacteria protect their own DNA from endonucleases?
Methylation
What is another name for the positive electrode?
Anode
What are the requirements for DNA gel electrophoresis?
gel: matrix for fragment separation
buffer: allows DNA to maintain charge
power supply: generates charge difference across gel
stain
How do we manufacture insulin?
take mRNA for proinsulin from pancreas use reverse transcriptase create cDNA of proinsulin join to a plasmid (recombinant DNA) inject into bacterium production of proinsulin occurs
What is the sequence of events for PCR?
heat to 95’C: break H bonds
lower to 55’C: allow DNA primers to anneal
heat to 72’C: optimum temp for Taq DNA polymerase
How many primers do we need for PCR?
2
They define the region we want to amplify.
Which direction does polymerase work?
5’ -> 3’
What is different with protein electrophoresis to DNA?
gel is upright with wells at the top
electrodes are placed dependent upon intrinsic protein charge
What is the major protein constituent of serum?
Albumin
How does SDS-PAGE work?
proteins separated purely on the basis of size
denature proteins so that they have a uniform negative charge
movement towards positive electrode based just on size
What is isoelectric focusing and how does it work? (IEF)
proteins separated on the basis of charge
stable pH gradient established
proteins migrate until they reach pH = to their pI
at their isoelectric point they have no net charge so stop migrating
What is 2D PAGE and how does it work?
2 step separation of proteins
use isoelectric focusing first to separate by charge
followed by SDS-PAGE to separate by size
allows separation of complex mixtures
Define proteomics
Analysis of all proteins expressed from a genome
How do we perform proteomics?
digest protein with trypsin
perform mass spectrometry
generate list of peptide sizes
use database to identify proteins
What are the features of polyclonal antibodies?
produced by many B lymphocytes
multiple different antibodies
specific to 1 antigen each
have multiple epitopes
What are the features of monoclonal antibodies?
produced from 1 B lymphocyte
1 identical antibody
specific to 1 antigen and 1 epitope
What do we detect with Western Blotting and how does it work?
proteins detected, proteins separated by SDS-PAGE, bound to more solid matrix, incubated with primary antibody for protein interested in, add enzyme linked secondary antibody that binds to primary that contains a marker
What is ELISA and how does it work?
Enzyme-linked immunoabsorbant assay
antigen coated wells
specific antibody binds to antigen
enzyme-linked antibody binds to specific antibody
substrate is added and converted to a coloured product by the enzyme
rate of colour formation is proportional to amount of specific antibody
How do radioimmunoassays work?
same way as ELISA
uses radiolabelled primary antibodies
What would you look for after an MI and using which technique?
creatine kinase and cardiac troponin 1
ELISA
What techniques can we use to analyse DNA at a chromosome level?
karyotyping and FISH
Why is a mismatch of a probe at the 3’ end more dramatic?
DNA polymerase cannot bind and extend so there will be no PCR product
