Albert Bolhius: Recombinant Dna Technology Flashcards
What are e coli and recombinant plasmids mixed with to allow plasmids to be taken up?
Mixed in the presence of CaCl2 and heat pulse
Somehow this creates pores and allows plasmids into the E coli
What bacteria is initial cloning stages usually done in?
E.coli
Recombinant plasmids mixed with it, and E coli them cultured
In PCR, what temp is desaturation of DNA strands (separation) done at?
94 degrees, takes around 30 seconds
What temperature is annealing with primers done at?
Cooled to 55-65 degrees from 94 degrees that was needed for desaturation, primers need this cooler temp to anneal, only takes 30 seconds
What temp is elongation with DNA polymerase done at?
72 degrees
DNA polymerase is thermostable
Takes 1 min per kilo base made (per 1000 bases)
When forming a DNA library, what vector is sometimes used? Why?
Phage ʎ
These can accommodate larger fragments than plasmids
But plasmids may still be used
What are the four ways to screen genomic libraries? Tricky to remember!!
Protein activity screening. ONLY works if activity of protein is easily measured.
Functional complementation: just remember you’re using strands of DNA you know are complementary to the gene you’re looking for that’s somewhere in the DNA library!
Hybridisation using DNA labelled probes, need info on gene sequence of interest to make the probe.
Immunological screening, using antibodies that bind proteins of interest.
What’s DNA sequencing used for?
Used to determine the order of nucleotides in DNA, ie determine the SEQUENCE
Used for:
Identifying sequence for drugs to target
Identifying mutated sequence that cause disease
Checking cloning experiments have worked propley
Understanding how genes function
How is DNA sequencing carried out?
A, T , C and G REACTIONS occur, whereby your sequence is cut/ fragmented at each one of these bases!
This forms every fragment possible
These fragments ran through sequencing gel (DNA electrophoresis)
Smaller molecules move fastest, to end of plate, larger molecules move slower.
Read sequence from bottom of plate!
The A,T,C and G reactions in DNA sequencing, where the sequence is fragmented at each base, can be done chemically or enzymatically.
What is the name of the chemical method?
What is the name of the enzymatic method?
Chemical: Maxam and Gilbert
Cleavage of bases here uses chemical reactions
Enzymatic: Sanger sequencing
How does Sanger sequencing work? What’s the enzyme used?
The enzyme DNA polymerase incorporates ddNTPs (deoxynucleotides) into the sequence (chain)
Whenever one of these ddNTPS (eg ddATP, ddGTP, ddCTP) is incorporated into the chain, the reaction stops and a FRAGMENT forms, as the chain is terminated
Eventually get a fragment at every single base
Run these through gel electrophoresis, seperates fragments and you can read the sequence from the bottom.
With the Sanger sequencing method, how can the nucleotides ddATP, ddGTP, ddCTP ddTTP be labelled? (hint: 2 options)
Radioactive labelling using radioactive isotopes attaching to diff nucleotides.
Fluorescent labelling, each of the four labelled with four diff colours . A computer reads the sequence.
What is bioinformatics?
You are translating a DNA sequence into its amino acid sequence.
You then compare the sequence translated with a data base (BLAST-N, BLAST-P, BLAST-X)
Then you can compare sequences to for eg find mutated genes, or identify similar sequences in diff organisms.
In bioinformatics, when translating the sequence, how many different reading frames are possible?
3 per strands; 2 strands so 6 reading frames in total.
Only one of these reading frames usually contains correct gene.
Start reading cram with start codon ATG
When viral DNA gets into one of our cells, enzymes will digest this DNA. Why don’t these digest enzymes digest out own DNA?
Some enzymes modify our own DNA by adding a methyl group.
When the methyl group is present the restriction enzymes won’t be able to cut the DNA.
Where do restriction enzymes cleave? What are these sequences called?
Cleave at palindromic sequences (reads the same forward and backward) this can create blunt or sticky ends
Does the EcoRI leave blunt or sticky ends?
Sticky ends
Enzyme from e coli
Restriction enzymes cut,
What enzymes paste?
Dna Ligase
It’s an ATP dependent enzyme
Which has better resolution, polyacrylamide gel or agarose gel?
Polyacrylamide
Agarose gel easier to use, allows you to run through bigger fragments
What is the typical DNA digest mixture composed of that you’d run through gel electrophoresis for 60 min at 60-129 volts?
DNA Water Concentrated buffer Restriction enzyme (also need a molecular weight marker, and after gel has been run, need to stain with a fluorescent marker and visualise under UV light)
When a DNA digest using restriction enzymes has been ran on a gel electrophoresis, and fragments have seperated, how do you determine size on fragments?
Bands visualised under UV light
Marker bands, of a known size, are held against the bands on your gel fro reference. You can read off sizes.
For a ligation reaction, what does the mixture require?
Vector
Insert (fragment produced by your restriction enzyme)
Buffer
Water
Ligase enzyme
Restriction enzyme to cut vector (SAME restriction enzyme used to cut fragment!)
When using dna ligase, The ends of the DNA fragment and ends of the cut vector must be complimentary, how’s this achieved?
Used the SAME restriction enzyme to cut the vector and make the DNA fragment inserts.
It will locate the same restriction site and therefore cut producing complimentary ends
What kind Of bonds does the Ligase form between the insert and the vector?
Covalently linked
by 3’-5’ phosphodiester bonds of the DNA
Can two different fragments digested by two different restriction enzymes possibly Ligate together?
Yes they can
Providing the sticky ends are the same sequence
Two different restriction enzymes may cut the same sticky ends
