Diagnostic Flashcards
What does genetic screening allow us to do
CPD
Causation
Prediction
Determine the type of cancer n trt
What’s the key process to genetic analysis
Hybridisation
Of a short piece of prob seq to complementary DNA/RNA
FISH method
DNA prob with fluorescent
Binds to the target DNA seq
Target seq retain fluorescent
Detect GROSS change to gene and chromo eg deletion / duplication
FISH is a diagnostic tool used for
Gene amplification - breast cancer HER2
Gene translocation - CML BCR-ABL
Gene inversion - NSCLC, EML4-AKL fusion
Can PCR amplifies whole genome?
No too larger w billion bps
Only allows dna from a selected region of genome to be amplified
And provided that the seq is already known
What do u need to do PCR
Template - has to be DNA
primers - 18-22 bp, complementary to 3’ end of each template to amplify
DNA polymerase - replicates from 3’ end of each primer
4 dNTP
Step 1 PCR dénaturation at what temp for how long
94C for 2 mins
Step 2 PCR annealing what temp for how long
Cooling of dna to 30-65 C
In presence of primers
30 sec
Depends on melting temp of primer
Step 3 PCR DNA synthesis what temp for how long
What polymerases are the choices
65-75
Use thermos table enzyme
5 mins
Taq . Pfu - can proofread
The second cycle of PCR happened at what temp for how long
94C for 30 SEC
Describe reverse transcriptase PCR
A method to amplify a RNA of known seq
Initial conversion from rna to dna by rna transcriptase
use poly T primer bind to poly A tail in rna
Synthesize cDNA
Rnase dégrade rna
Use cDNA, use DNA polymerase form ds dna
…
How are PCR products detected
Slab gel electrophoresis
Capillary gel elctrophoresis
Describe SGE
Slab gel electrophoresis - use ethidium bromide to stain PCR DNA product (or primers w fluorescent/ conjugated enzyme) - product into agarose gel plate - visualises by UV light - apply electricity - dna moves due to po4 3- backbone - ‘multiple products can be detected on single gel is mw differs Multiplex rxn
Describe CGE for PCR product analysis
Sample pass through a capillary
Separated by size / mass
Laser used to detect fluorescence
PCR can be used to detect …
rely on
Point mutation (bp) / SNP (1 base) Deletion Insertion Eg NSCLC Rely on RFPL, or if working at all
What’s is RFLP
Restriction fragment length polymorphism
Due to mutations in creation/ deletion of restriction site on dna seq
Pcr products will have RFLP when incubated w restriction enzyme
What’s the most common gene mutation in NSCLC
How does it give RFLP
L 858 R
Restriction enzyme cannot cleave the genes at the original restriction site due to mutation — give a full length band for the whole product
What’s allele specific PCR rely on I order to detect Point mutation
Rely on the correct base at 3’ end of primer
Important starting point for synthesis
Designed single base (3’ end of primer) that is NOT complementary to mutant base (PM)
So no amplification of gene if mutated
In real time PCR which strand does the TaqMan prob bind at what site
On forward strand
Minor groove binding site
How does real time PCR allow you to do and how does it work
Allow you to quantify the amount of dna in the sample to start with, also called q-PCR
Oligo seq bind to forward strand
DNA pol’s 5’ nucléase dégradé prob
Release F reporter
What is cycle threshold in real-time PCR
The number of cycle it takes for you to be able to detect the fluorescent generated by cleaving taqman prob
