BIOL 114 Flashcards
(70 cards)
How do you obtain DNA without introns?
You take mRNA from lysed cells and use reverse transcriptase to make cDNA and Degrade the RNA with RNAse H so you can synthesize a whole DNA using primers, nucleotides and DNA polymerase.
How are genes cloned in bacteria?
A fragment of DNA is inserted into a circular DNA vector which is inserted into a bacterium (usually) which replicates the vector and the cells
How do you identify bacteria that have taken in a circular DNA vector?
The vector contains an antibiotic resistance gene and so any bacteria that survive said antibiotic will contain the DNA
What are restriction enzymes?
Enzymes that cleave DNA at specific sites in the nucleotide sequence
What does DNA ligase do?
It joins together DNA fragments by ligating the phosphodiester backbone
What are the conditions that make bacteria uptake DNA more efficiently?
Soaking in ice cold salt solution and then incubated at 42 degrees for 2 minutes
How do you minimise the chance of vector religation in bacteria?
Using phosphatase to remove 5’ phosphate groups
How do you identify cells that contain recombinant DNA after the antibiotic treatment?
Using a marker gene such as LacZ’ which makes an enzme (Beta-galactosidase) that converts X-gal into a blue coloured product.
You then put the gene of interest inbetween that gene so that if a blue product is produces then the target gene is not present to interupt so you can get rid of any blue colonies.
In electrophoersis how does the % agarose gel affect the resolution of DNA molecules that can be seperated?
Using higher percentages increases the resolution (smaller base pair sequences can be seperated)
If a healthy person prpduces 2 strips on electophporesis and a diseased person only produces one when cut with the same restriction enzyme what can you tell by this?
That a mutation has occured where the restriction enzyme would normally cut
How is strand synthesis stopped in sanger sequencing?
ddNTP is added which terminates the synthesis.
What sequencing technique was used to obtain the first human genome draft?
The chain-termination (sanger) method
What techniques are used for measuring mRNA?
Northern Blotting
RT-PCR
Microarray (genechip) techniques
Since all mRNA have a poly-A tail what does a poly-dT primer do when reverse transcriptase added?
It converts all the mRNA in the cell into cDNA unless a specific primer is added.
How is analysis of mRNA done to find gene expression (both current and previous methods)
Previous method: Microarray with flouresence labeled cDNA
Current: RNA sqeuencing which allows large groups of genes to be studied
How does nanopore sequencing work?
Nanopores embedded in synthetic membranes have ionic current measured as they pass through and when DNA passes through these nanopores it disrupts the current in specific ways based on the base pair passing through therefore this can be used to determine nucleotide sequence
Whats the difference between genomic libraries and cDNA libraries?
Genomic contains non-coding regions and is used to reconstitute genomes to study gene function
cDNA libraries only contains exons from mRNA and is used to reconstitute proteomes to study protein structure
What is hybridization?
Locating cDNA clone using a nucleic acid probe that consists of 100-1000 bases specific to the target cDNA that is labelled so it can be identified.
What types of probes are used in hybridization?
Homology probe: Corresponding DNA sequence from a related organism so DNA is almost identical)
Degenerate oligonucleotides: Degenerated DNA sequence from a known protein sequence. This can either be multiple labelled oligonucleotides for each option or using inosine-based artificial nucleotides where there is degeneracy in the code
What labels are used in hybridization?
Radioactive labels (phosphorus)
Non-radioactive labels (more complex to detect uch as DIG-specified antibody detection)
What is immunodetection?
Using antibodies to recognise protein produced by target cDNA
Proteins are transferred to nitrocellulose membrane (every protein binds to it) and add the antibodies to recognise them (after blocking any free nitrocellulose spots).
What is an expression system for recombinant proteins and what are some considerations to make when choosing one?
The host cell where you are making your recombinant proteins
Considerations:
Size
Solubility (membrane proteins wont be easily recovered from bacteria)
Post-translational modifications (bacteria can’t do this)
What can be optimised in E. coli expression?
The strain
The plasmid vector
The coding cDNA sequence inserted
How does optimising codon usage improve translation rate?
Degeneracy leads to codon bias where some codons are expressed more frequently so using these will improve rate and yield
