PCR and qPCR Flashcards
PCR used to
used for making many copies of DNA
PCR is good because
- it is quick
- can easily choose which bit of DNA to amplify
- can be used to detect specific DNA sequences or to change them
if we think someone may have a virus
we can use PCR to detect presence or absence
why we use PCR for viral DNA
viral genomes are only a few thousand BP in length, human genome is 3 bill BP- hard to detect such small piece–> therefore PMR is integral
viral DNA will be diff to
host cell e.g. identifies sequences of 18-20 bp in viral dn, specific and not found in human genome
specific sequences found
at the end of the top strand and the other end of the bottom strand and complementary oligonucleotides will be produced (primers)
process of PCR
1) heat to 96- denature- separate strands
2) primers added- cooler (60)- and attach in-between DNA
3) heated back to 72- extension- DNA Polymerase attaches to primers (only works when added to double strand)
4) DNA is amplified
5) this cycle is repeated
how many times is the PCR cycle repeated
30 times
each cycle..
doubles the amount of DNA
PCR is known as a
chain reaction
DNA can be separated based on
its size and how far is moves on agarose gel
viral DNA will only have
300bp
qPCR stands for
quantitative DNA
what is qPCR used for
to quantify the amount specific DNA sequence
- quantifying expression levels of mRNA
example of how qPCR can be used
e.g. if we look at a normal cell vs a cancerous cell- we can look if RAS is being over expressed in 1 more than the other
what is used to quantify mRNA
rt-PCR
PCR only works on
DNA- so reverse transcriptase (mRNA- cDNA) is used to purify mRNA from normal and cancer cells
qPCR process
1) add RT to mRNA to produce cDNA
2) cDNA could be PCRs if cloning or for qPCR
3) the DNA produced is measured by a dye that fluoresces when bound to DNA
4) primes specific to RAS DNA used
5) presence in PCR is SYBR green dye which binds to double stranded DNA and fluoresces
6) the more starting material you have the more double stranded DNA you will have and the more it will fluoresce
7) we need a control- take our cDNA mix and do separate qPCR reaction with primers for a gene whose expression ‘we think’ doesn’t change.
why do we need a control for qPCR
take our cDNA mix and do separate qPCR reaction with primers for a gene whose expression ‘we think’ doesn’t change. This gene is often GAPDH.
should really test a panel of genes
under diff conditions
