Lec 16 - Intro to molecular diagnosis

we can use : Restriction enzymes DNA gel Electrophoresis PCR (polymerase chain reaction)

Protein electrophoresis Immunoassays Enzyme assays

Southern hybridisation microarray PCR variations

DNA Sequencing

Karyotyping FISH

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

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

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

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

• 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

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

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

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

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

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

Radioimmunoassay - radiolabeld antibody continuous - spectrophotometry or chemoluminesence Discountinuous - radionactivity or chromatography this slide is not very improtant

look for metabolic disorders in tissues look to diagnose disease in serum enzymes to diagnose a myocardial infraction - we measure cardiac troponin and CK MB which will be very high if an MI has occured