Diagnostic

Question 1

What does genetic screening allow us to do

Answer 1

CPD
Causation
Prediction
Determine the type of cancer n trt

Question 2

What’s the key process to genetic analysis

Answer 2

Hybridisation

Of a short piece of prob seq to complementary DNA/RNA

Question 3

FISH method

Answer 3

DNA prob with fluorescent
Binds to the target DNA seq
Target seq retain fluorescent
Detect GROSS change to gene and chromo eg deletion / duplication

Question 4

FISH is a diagnostic tool used for

Answer 4

Gene amplification - breast cancer HER2
Gene translocation - CML BCR-ABL
Gene inversion - NSCLC, EML4-AKL fusion

Question 5

Can PCR amplifies whole genome?

Answer 5

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

Question 6

What do u need to do PCR

Answer 6

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

Question 7

Step 1 PCR dénaturation at what temp for how long

Answer 7

94C for 2 mins

Question 8

Step 2 PCR annealing what temp for how long

Answer 8

Cooling of dna to 30-65 C
In presence of primers
30 sec
Depends on melting temp of primer

Question 9

Step 3 PCR DNA synthesis what temp for how long

What polymerases are the choices

Answer 9

65-75
Use thermos table enzyme
5 mins
Taq . Pfu - can proofread

Question 10

The second cycle of PCR happened at what temp for how long

Answer 10

94C for 30 SEC

Question 11

Describe reverse transcriptase PCR

Answer 11

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
…

Question 12

How are PCR products detected

Answer 12

Slab gel electrophoresis

Capillary gel elctrophoresis

Question 13

Describe SGE

Answer 13

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

Question 14

Describe CGE for PCR product analysis

Answer 14

Sample pass through a capillary
Separated by size / mass
Laser used to detect fluorescence

Question 15

PCR can be used to detect …

rely on

Answer 15

Point mutation (bp) / SNP (1 base)
Deletion 
Insertion
Eg NSCLC
Rely on RFPL, or if working at all

Question 16

What’s is RFLP

Answer 16

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

Question 17

What’s the most common gene mutation in NSCLC

How does it give RFLP

Answer 17

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

Question 18

What’s allele specific PCR rely on I order to detect Point mutation

Answer 18

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

Question 19

In real time PCR which strand does the TaqMan prob bind at what site

Answer 19

On forward strand

Minor groove binding site

Question 20

How does real time PCR allow you to do and how does it work

Answer 20

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

Question 21

What is cycle threshold in real-time PCR

Answer 21

The number of cycle it takes for you to be able to detect the fluorescent generated by cleaving taqman prob