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PMR > techniques > Flashcards

techniques Flashcards

1
Q

what does PCR Do

A

amplifies specific sequence of DNA

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2
Q

qualities of PCR

A

specific

very sensitive

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3
Q

uses of PCR

A

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

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4
Q

process of PCR

A

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

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5
Q

what does qPCR do

A

quantifies a specific DNA sequence in a sample

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6
Q

qualities of qPCR

A

very sensitive

accurate and reliable

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7
Q

uses of qPCR

A

comparing amounts of DNA
quantifying a viral load
compare amounts of mRNA (qRT-PCR)

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8
Q

process of qPCR

A

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

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9
Q

analysis of qPCR

A

absolute quantification

relative quantification

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10
Q

absolute quantification

A

analysis of qPCR
measures level of gene expression in 1 sample
compares Ct value of sample of interest with standard curve

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11
Q

relative quantification

A

measures relative change in gene expression between 2 samples

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12
Q

calculating relative quantification

A

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

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13
Q

importance of reference gene in relative quantification

A

ref gene will be expressed at the same level for each sample so allows comparison of unknown gene

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14
Q

standard curve for absolute quantification

A

plot Ct values against log(DNA concentration)

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15
Q

quality of RNA sequencing

A

un-biased

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16
Q

what does RNA sequencing do

A

quantifies how much of each mRNA you have from each gene in a sample

17
Q

uses for RNA seq

A

compares gene regulation or expression in a particular disease or in response to a particular stimulus

18
Q

example of RNA seq

A

comparison of mRNA expression under hypoxic conditiions compared to normoxia

19
Q

process of RNA seq

A

quantifies RNA
converts RNA to cDNA
carries out parallel sequencing to identify each molecule

20
Q

genome sequencing

A

parallel DNA sequencing but on a bigger scale

21
Q

uses of genome sequencing

A

allows identfiaction of spontaenous mutations

22
Q

purpose of DNA sequencing

A

determines the base sequence of a stretch of DNA (around 750 bp)

23
Q

uses of DNA sequencing

A

detection of mutations
identification of pathogens
identification of specific genes

24
Q

process of DNA sequencing

A

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)

25
Q

what does GWAS do

A

uses SNPs to link genetic variation to disease

26
Q

uses of GWAS

A

helps understand pathways involved in complex diseases

allows assessment of an individuals disease risk

27
Q

what does trio-analysis do

A

identifies disease-causing spontaneous mutations

28
Q

how does trio-analysis work

A

screen the parents
screen the child
if the child has any genes that arent in parents genomes - it is a spontenous mutation

29
Q

use of trio-analysis

A

identifies mutations causing rare genetic diseases

30
Q

knock-out models

A

inactivation/disruption of a specific target gene

commonly use mice

31
Q

use of knock out

A

assess function of a gene

create a disease-model

32
Q

what is CRISPR

A

genome editing technique

allows user-targeted disruption or alteration of mammalian genome

33
Q

uses of CRISPR

A

creating knock-outs

therapeutic repair of mutant genes

34
Q

what does a western blot do

A

uses a specific antibody to detect and quantify a particular protein in a sample

35
Q

uses of western blotting

A

detect changes in protein level

detect disease specific expression

36
Q

what does a genetic screen do

A

randomly mutate genes (at a low level) in various model oragnisms
see if function is affected

37
Q

uses of genetic screens

A

identify genes important for a particular physiological or developmental process

38
Q

what is statistcal analysis of RNA sequencing looking for (compare to known gene database)

A

looking for over-representation of genes in known pathways

39
Q

annotated gene database

A

every gene in human genome has pathways, associated genes and proteins involved written next to them

PMR (9 decks)

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