Lec 16 - Intro to molecular diagnosis Flashcards
What are the techniques to analyse DNA at a gene level ?
we can use :
Restriction enzymes
DNA gel Electrophoresis
PCR (polymerase chain reaction)
How do we analyse proteins ?
Protein electrophoresis
Immunoassays
Enzyme assays
Advanced Molecular Techniques :
How do we analyse DNA at the gene level ?
Southern hybridisation
microarray
PCR variations
Advanced Molecular Techniques :
How do we analyse DNA at the nucleotide level ?
DNA Sequencing
Advanced Molecular Techniques :
How do we analyse DNA at the chromosome level ?
Karyotyping
FISH
How do we use restriction enzymes to analyse DNA at a gene level
Uses endoneucleases produced by bacteria
these will recognise and cut specific sequences within DNA
ECOR1 - Ecoli - recogs and cuts GAATTC
can be used in conjucntion with DNA gel electophoresis
allows us to identify and cut parts of a genome
We can see the size of DNA fragments, investigates mutations such as sickle cell disease
DNA fingerprinting
clone DNA in PCR
How do we use DNA gel electrophoresis to analyse DNA at a gene level
DNA fragments that have been cut are put into a gel, with positve and negative electrodes
negative DNA will travel towards positive electrodes
smaller fragments will travel further due to size restrictions
DNA will be stained (ethidium bromide) to make it visible
what is Gene Cloning ?
why do we use it ?
use plasmids (circular DNA found in bacteria) palsmids can replicate independently
- use restriction enzmyes to isolate gene of interest
- insert this gene into the plasmid
- introduce recombinant DNA into suitable host cell - e coli
- DNA multiplies
- identify and isolate the clone with DNA of interest
the why :
we do this to make useful human genes such as insulin
find out what genes do
genetic screening - huntingtons, cystic fibrosis
use the example of insulin for gene cloning
isolate mammilian proinsuling mRNA - from pancreas
use mRNA as this is active and has no introns
use a reverse transcriptase to produce pronsulin cDNA
join to plamsid to make recombinaint DNA
infect ecoli
Bacteria replicates - then harvest produced human insulin
explain the process of PCR
- Heat soultion to 95 C - this causes the DNA to denature into 2 single strands
- cool to 55 C - this allows forward and reverse primers (for the section we want) to anneal - primer annealing to DNA strand
- Heat to 72 C - this allows a Taq Polymerase enzyme (thermostable at high temps) to catalyse Primer extension
- Repeat process around 30 times getting exponential growth - to produce billions of copies
why use PCR ?
amplify a specific DNA fragment
investigate single base mutations - sickle cell
investigate small deletions of insertions - cystic fibrosis
investigate genetic relationships - DNA profiling
we need to monitor what happens in PCR
we use it in conjunction with another technique
explain protein electrophoresis
proteins are charged will move towards cathode or annode
we cans epreate proteins based on size and charge
works the same as DNA electrophoresis - uses a porus gel ect
explain SDS-Page
allows for the separation of complex mixtures of proteins
good for diagnosing disease states in different tissues - look at all proteins in a tissue sample at once and compare
we can see if a protein has been up regulated or down regulated - ie in a tumor
treat sample with a reducing agent - gives all proteins same negative charge - will move towards positive electrode
apply charge over porus gel and proteins will separate by size and charge - smaller less charge moves further
use a staining technique to make proteins visible
antibodies bind to specific antigens
there are polyclonal antibodies - multiple antibodies specific to one antigen - recog many epitopes
monoclonal antigens - 1 antibody specific to 1 antigen - recog 1 epitopes
western blotting uses antibodies , How ?
we can use antibodies to label a specific protein
we bind a primary antibody to a proteins
we bind a secondary linked antibody (if it bound to protein) - produces an immunoblot
what are enzyme assays (ELISA), how do they work ?
antigen we want to target / see if it present
our specific antibody binds to the antigen
an enzyme linked secondary antibody binds to specific antibody
we adds substrate - enzyme converts to coloured product - rate of colour formation is proportional to amount of specific antibody
we can measure the rate of an enzyme catalysed reaction - compare to normal rates - ie if there are liver enzymes in the serum - we have liver damage
we can use this to measure conc of proteins in solution - insulin, TSH, Cortisol
