Fergus - Applications of PCR Flashcards
List some applications of PCR
(8)
Amplification of known sequences
Amplification of sequences using amino acid information
Reverse Transcription (RT-PCR) using RNA template
Mutation detection -> product sizing, ARMS PCR, SSCP, sequencing
Linkage analysis
Subcloning of sequence
Arbitrarily primed PCR
Real Time PCR
Give an example of how PCR can be used for the amplification of known sequences
Amplification of the insulin gene so it can be produced by bacteria
Give an example of how PCR can be used for the amplification of sequences using amino acid information
(4)
Used when we don’t know the gene
We can design primers from amino acid sequences
We can translate the list of amino acids into a possible DNA sequence - taking into account the different ways of making each amino acid e.g. TGT or TGC for cystine (primer contains 50% TGT and 50% TGC)
These primers we create are called degenerate primers -> they can be used for both amino acid variables
What is reverse transcription PCR?
(5)
This is where we start with RNA (which cannot be used be used as a PCR template)
We have to convert the RNA into DNA
The mRNA has a polyA tail
Oligo primers are used -> they bind on and reverse transcriptase adds nucleotides on (dNTP)
This gives us a single strand and allows us to amplify the entire coding section of genes
What is mutation detection PCR?
(5)
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
Enzymes are used to cut the sequence -> normal is cut, carrier has some uncut and some cut
What is amplification resistant mutation system (ARMS) PCR?
(4)
Create two sets of primers
Primers will amplify the normal sequence and another mutated primer sequence
e.g. in CFTR -> one primer matches GAA and the other matches GTA (mutation)
Normal primers sequence the normal gene, mutated primers match the mutated sequence
Normal primers won’t amplify the mutated sequence, mutated primer wont amplify the normal sequence
What is a multiplex ARMS PCR?
(2)
ARMS PCR but for different mutations at the same time
e.g. in PCR for CF -> normal primers and four different mutated primers
What is single strand conformation polymorphism?
(3)
This is where we look at each exon individually
We use SSCP gel
If something comes up as mutated we than use SSCP to sequence the exon and find out what exactly the mutation is
List the different types of mutation detection PCR
Product sizing
ARMS PCR and multiplex ARMS
SSCP
What is linkage analysis?
(3)
Allows us to follow chromosomes and their mutations through a family
It allows us to find the rough location of the gene relative to another DNA sequence called a genetic marker
We can identify what chromosomes have caused a disease(what chromosome the mutation is on)
What is arbitrarily primed PCR?
(3)
Also called random amplified polymorphic DNA (RAPD)
We design short random primers and apply conditions that will promote non specific products
Useful in microbiology to identify bacteria - as we will get a series of non specific products however if we repeat this we will get the same results -> used to identify bacteria
What is real time PCR?
PCR where we can detect products as they are being generate using fluorescent labelling
Dependent on fluorescent detection of PCR products during cycle
DNA is split into single strands, fluorescent dyes are attached,
