week 3- studying gene expression Flashcards
do all cells in the body share the same genome
yes, just expression of genes is different in other/ different cells
what are some techniques for protein analysis
· SDS-polyacrylamide gel electrophoresis and western blotting
· Immunofluorescence / immunohistochemistry
Fusion proteins
what is a restriction enzyme that gives rise to a) blunt end 2) sticky end
Some restriction enzymes cut blunt (haelll)or stick ends (ecoRI HindIII)
why are sticky ends better
· The sticky ends are better because they leave ‘overhang’ which help to join the DNA back together through complimentary base pairs. The re-joining of DNA is important in DNA cloning
how does gel electrophoresis work
· Separated DNA from size
· Different restriction enzymes cut at different sites, thus the fragments are separated
· The negative DNA will migrate to the postive electrode
The lower bands are closer to the positive electrode
what are the 3 steps of recombinant DNA
1) transform e. coli with the recombinant plasmid where some are taken up, none are taken up
2) plate bacteria with antibiotic resistance
3) Isolate multiple individual colonies and amplify each colony in a separate flask.
what is the ori sequence
An origin of replication site which promotes replication in bacteria
what is AMP and what is it purpose
For bacterial resistance to ampicillin, this ensure that only bacteria which contain the plasmid will grow on culture plates containing ampicillin
what is GFP
green flourencet protein so we can see the cultures
steps of PCR
1) heat DNA to 96 degrees to seperate the 2 DNA strands by denatureing the hydrogen bonds
2) sample is cooled to anneal the primers
3) Taq polymerase is added to synthesise a complementary DNa sequence
4) the process is completed until stopped it runs out of bases
what is one application of recombinant DNA and molecular biology
HIV
what are the tools to use molecular biology to understand the HIV virus
- blood sample with a person infected with HIV
- extract the RNA from the HIV particle in the plasma of the infected person
- use reverse transcription and PCR of the cDNA
- put this into gel electrophoresis agaisnt a person who is not infected to see the BP of the virus
- put the sample in a plasma vector (recombinate DNA) to overexpress the protein
what can a genomic DNA library be used for
A genomic DNA library can be used to determine the nucleotide sequence of an entire genome.
what can a cDNA library contain
A cDNA library contains clones at a frequency that mirrors the frequency of mRNA molecules in tissues.
what is the overall simplified version for a cDNA library
We can isolate DNA and fragment into plamids to create a library
what are the steps to make a genomic library
- double stranded DNA is cleaved with restriction enzymes, crating millions of DNA fragments
- DNa fragments are inserted into plasmids
- these fragments are introduced into bacteria which creates a genomic bacteria
what is RNA-seq
RNA sequencing
what are the steps for RNA-seq
- Extract and fragment RNA from cells/tissue/sample of interest
- Synthesise cDNA (convert mRNA to cDNA)
- Adapter ligation (add adapters to ends of cDNA fragments)
- PCR amplification of sequences of interest
- Select fragments of desired size
- Sequence
`