techniques Flashcards
what does PCR Do
amplifies specific sequence of DNA
qualities of PCR
specific
very sensitive
uses of PCR
detection of viral and bacterial DNA in a tissue sample
determine the size of a sequence between 2 primers
production of DNA genetic tools or DNA sequencing
process of PCR
identify specific sequence (around 20bp)
make specific reverse and forward primers
heat to 96 - denature - single stranded
annealing - cool to 60 - primers added
extension - add dNTPs - 72 optimum temp for DNA polymerase
repeat and separate using electrophoresis
what does qPCR do
quantifies a specific DNA sequence in a sample
qualities of qPCR
very sensitive
accurate and reliable
uses of qPCR
comparing amounts of DNA
quantifying a viral load
compare amounts of mRNA (qRT-PCR)
process of qPCR
if RNA - convert to cDNA
carry out PCR process to amplify
measure DNA by fluorescent labels which fluoresce when bound to DNA
plot fluorescence against number of PCR cycles
analysis of qPCR
absolute quantification
relative quantification
absolute quantification
analysis of qPCR
measures level of gene expression in 1 sample
compares Ct value of sample of interest with standard curve
relative quantification
measures relative change in gene expression between 2 samples
calculating relative quantification
carry out qPCR on the 2 samples and for the reference gene
calculate the difference in Ct values between either sample and ref gene
divide delta Cts by each other to give comparison
importance of reference gene in relative quantification
ref gene will be expressed at the same level for each sample so allows comparison of unknown gene
standard curve for absolute quantification
plot Ct values against log(DNA concentration)
quality of RNA sequencing
un-biased
what does RNA sequencing do
quantifies how much of each mRNA you have from each gene in a sample
uses for RNA seq
compares gene regulation or expression in a particular disease or in response to a particular stimulus
example of RNA seq
comparison of mRNA expression under hypoxic conditiions compared to normoxia
process of RNA seq
quantifies RNA
converts RNA to cDNA
carries out parallel sequencing to identify each molecule
genome sequencing
parallel DNA sequencing but on a bigger scale
uses of genome sequencing
allows identfiaction of spontaenous mutations
purpose of DNA sequencing
determines the base sequence of a stretch of DNA (around 750 bp)
uses of DNA sequencing
detection of mutations
identification of pathogens
identification of specific genes
process of DNA sequencing
carry out PCR to amplify
also add fluorescent-labelled terminator dNTPs
DNA fragments extend up to added terminator bases (lots of different sizes)
electrophoresis to separate fragments by size
as fragments pass through, terminator sequence fluorescs (different colour = differnt base)
