Fergus - PCR Question Flashcards
When asked about the principles of PCR, what do you need to write about?
What is PCR?
How PCR is carried out?
Diagram of PCR
What is PCR?
(5)
Method of DNA cloning (the isolation, purification and amplification of a specific DNA sequence)
Polymerase chain reaction
Method of amplifying DNA sequences in vitro
Exponential DNA replication process
Requires some knowledge of sequence to be amplified
What are the steps to PCR
(6)
Choose the sequence you want to amplify
Design complimentary oligonucleotide primers,
Denature the DNA,
Anneal primers,
Amplify clone using Taq Polymerase
Repeat process
How do you denature DNA?
By heating the reaction up to about 50-65 degrees Celsius
This breaks the hydrogen bonds between the two strands of DNA
Results in single strands of DNA
How do you anneal primers?
We can now anneal our primers to the single strands using the correct optimal annealing temperature
How do we extend our primers?
(3)
Add our Taq Polymerase along with dNTP mix
Increase temperature to 72 degrees Celsius for Taq to work
Repeat this cycle
List the six components of PCR
DNA template
Primer oligonucleotides
dNTP mix
Thermostable DNA polymerase
Buffer and additives
Write about the DNA template component of PCR
(3)
A sequence of DNA that contains the target of amplification
The sequence must not be degraded
Different examples:
Bacterial DNA
Viral DNA
Vector DNA
cDNA/Genomic Libraries
Products of reverse transcription reactions
Write about the primer oligonucleotide component of PCR
(6)
Synthetic DNA sequences
Complimentary to target sequence
Provide primer site for DNA polymerase
Generally 18-30 bp in length
Should not anneal to each other
Should anneal to complimentary sequences at approximately the same temperature
Write a note on the dNTP mix component of PCR
(8)
Components of DNA
There are four types of dNTP, one for each nitrogenous base, in a mix the four are found in varying quantities
Contains dATP, dCTP, dGTP, dTTP
Required by DNA polymerase for chain extension
Supplied at concentrations that will not limit DNA
Nucleotide analogues can be used to avoid DNA structure problems
Fluorescent labels can be added on e.g. fluorescein and biotin
Radioactive labels can be added on e.g. 32P and 35S
Write a note on the thermostable DNA polymerase component of PCR
(3)
Adds dNTPS onto the 3’ hydroxyl of an extending strand
Must be thermostable to be able to withstand the high temperatures of PCR
E.g. T7 (sequenase), Taq polymerase, Thermosequenase
Write a note on the buffer and additives component of PCR
The thermostable DNA polymerase is used to working in certain conditions e.g. in an organism where there is a certain pH and certain nutrients available
Therefore these environmental conditions must be mimicked in PCR for polymerase activity to work
Additives = Salt and Magnesium
Give four applications of PCR
ARMS
Product sizing
RFLP
SSCP
Write a note on ARMS PCR
(7)
Amplification Resistant Mutation System
Allele Specific PCR
Primers are designed to amplify only the normal or only the mutated sequence
Based on the 3’ nucleotide of the primer matching exactly the target or mismatched
2 PCR reactions are performed
Normal primer, Mutation primer and common primer
The normal primer will only work on a normal sequence and a mutated primer will only work on a mutated sequence
Write a note on product sizing
(4)
Used to detect a deletion or a base change
If 3 base pairs are missing the product is going to be three base pairs shorter (product sizing PCR)
If a G converts to an A it doesn’t change the size of a PCR product
It can only be used to detect mutations that have been previously identified
