molecular techniques Flashcards
(28 cards)
What are restriction enzymes?
endonucleases made by bacteria that cut at specific DNA sequences
What is gene cloning
isolate gene using restriction enzyme gel extract ligate into plasmid vector transform into bacteria identify and isolate
How does DNA electrophoresis
DNA negatively charged moves towards positive electrode
movement dependent on size of fragment
Explain PCR
heat 95 to denature
cool to 60 +/- 5 primers anneal
extension at 72 degrees
Explain protein DNA gel electrophoresis
similar DNA electrophoresis
movement dependent on size and charge of protein
density gives indication of number of molecules
what does sds -page stand for
sodium dodecyl sulphate polyacrylamide gel electrophoresis
What does SDS do
make protein linear remove charge
makes protein negatively charged
neg charge proportional to size of protein
as a result movement of protein on gel dependent only on size influence of charge removed
What dye is used in sds page
coomassie dye
How do you do protein identification
digest protein with trypsin
perform mas spec
generate lots if peptide sizes
use database of predicted peptide sizes for know proteins to identify
How can ab’s be used
target specific epitope/ antigen
explain western blot
transfer to nitrocellulose gel
apply primary ab
apply secondary enzyme linked ab
immunoblot
What does ELISA stand for
enzyme linked immunoabsorbent assay
Explain process of DNA hybridisation?
denature dsDNA to ssDNA
add labelled probe complementary to sequence of interest
identify usually using radioactive film
What is a southern blot
identify sequences of interest after gel electrophoresis
gel transfer to nitrocelluse/ nylon membrane
alkaline solution to denature dsDNA
add probe
detect
what is a northern blot
identify rna using dna probe similar to southern blot
Key features of probes
do not need 100% similarity to sequence to bind, more similarity = greater binding strength
do not need to completely align with sequence they are being used to distinguish
do not affect position of band that is detected
Explain sanger sequencing
use ddNTP of A/T/G/C which stop dna elongation due to missing 3’oh group
use one ddNTP in each tube with the normal dNTPs extension will be terminated at different points
run all sample on gel
read sequence off the gel bottom up
How can identify mutants with PCR
run differently
allele specific primers
how can you identify mutants with southern blotting
use allele specific probes
Explain the process of RT-PCR
reverse transcriptase pcr mRNA present if gene expressed use primer with long poly a tail bind mRNA then add reverse transcriptase to make cDNA add nuclease breaks down rna ssdna produced carrier out pcr as normal to amplify dsDNA molecule
What does microarray allow? what are the two types ?
genome wide analysis of thousands of genes
conditional gene expression
array comparative genome hybridisation
Explain process of conditional gene expression
abnormal vs normal cell
rna to dna (tagged fluoresecently ) using reverse transciptase
hybridised to microarray
read gene expression
explain the process of array comparative genome hybridisation
normal vs abnormal
dna extracted and tagged
mixed in equal quantities and hybridised to microarray
read fluorescence
work out ratios of different fluorescence
decrease–> deletion
increase–> duplication
only one fluorescent- only present in one cell not the other
What is a microarray
microscope slide with specific dna sequences dotted on it on specific regions
