Recombinant DNA Flashcards
Recombinant DNA strategy general steps?
(transferring gene for fluorescence from a mould into a cat?)
Extract DNA from mould
Fragment the DNA using restriction enzymes
Ligate into cloning vectors
Create library in E.coli
Identify correct gene
Transfer to expression/shuttle vector
Express in target organism/cat
Components of plasmid cloning vectors?
Contain ori: origin of replication so they can replicate in the E.coli
Restriction endonuclease sites for cutting.
And genes for antibiotic resistance to identify if the E.coli contains the plasmid cloning vector and if the plasmid contains the ligated genes.
Or just an ampicillin resistance gene for selection of plasmid containing E.coli colonies and a lac operon with restriction sites for blue/white screening selection of ligated plasmids.
How does blue white screening work using plasmids containing Lac Z gene (actually gene for only one monomer of Lac Z enzyme) and addition of X-gal?
Lac operon deleted from bacterial chromosome.
When present, the lac Z enzyme converts chemical, X-gal into a blue colour.
Blue colour indicates lac Z gene intact.
When restriction enzyme allows ligation of gene into the middle of lac operon, lac Z is not present and so colonies with ligated gene stay white!
What is a phagemid?
A plasmid that contains a separate origin of replication (e.g. M13 ori) that produces a single stranded DNA copy of the plasmid packaged in a virus particle which could be useful for a variety of lab purposes including purification of sample.
What are the alternatives plasmid cloning vectors and why might you need them?
Plasmid cloning vectors are small, can only handle 10 - 20kb. so other bigger vectors are required for bigger genes or multiple genes.
Lambda bacteriophage or cosmids 50kb**, **plasmids containing cos packaging site from Lambda (like M13, packages plasmid into virus particle)
Artificial chromosomes: Bacterial, Yeast or Mammalian (not preferable as unstable, as so big, complicated) 600kb
How to fragment DNA after extraction?
Physical methods such as Sonication or Shearing that randomly fragment DNA.
Restriction endonucleases (8,6,4 cutters cut at specific sites) (8s less frequently, 4s more frequently) (ideally leaving sticky ends) (6 cutters are best, cut approx every 4kb, genes are about 1kb, 8s too big to fit cloning vectors, 4s too small segments to get a whole gene!) (or a partial digest with a 4 cutter)
What is the point of a 4 cutter restriction endonuclease partial digest?
4 cutters cut approx every 250b, genes are approx 1kb.
Partial digest involves using suboptimal conditions for digestion so 4 cutters don’t cut at all restriction sites, leaving a great variety of larger fragments!
(better than a 6 cutter because a 6 cutter could always have a restriction site within the gene!!)
How to ligate cut sequences into plasmids? (or other cloning vectors)
cut plasmids with same restriction endonuclease to give end
Use T4 ligase enzyme that uses ATP to ligate cut ends. AMP attaches to 5’ phosphate and nucleophilic attack of 3’ OH group on P-P forms new phosphodiester bond.
(keep large excess of inserts relative to plasmids to prevent plasmids re-ligating, or use phosphatases on plasmids)
How to prevent plasmid re-ligation? (promoting ligation of insert into plasmid)
Keep insert in large excess.
Or add phosphatases to broken plasmid solution. Removes AMP from phosphates on plasmids so their ends can’t re-ligate!
This means only one end of each insert-plasmid ligated strand can be ligated properly. This is good enough/stable enough for insertion into E.coli, who will fix the strands properly!
How to get plasmid vectors into E.coli (or other bacterial cells)?
Add Calcium chloride CaCl2 or use **electroporation **to make cells porous!
Each bacterial cell will only take up one plasmid!
What determines the number of clones/clonal colonies needed to ensure(/achieve a desired probability) ligated vector containing the desired gene is in one? (included in the library)
The size of the genome of the organism from which the DNA was extracted (the bigger the genome, the more colonies required, [so when too big a genome, use transcriptome!])
The size of the insert. (size of insert ligated into vector, bigger insert = fewer colonies needed)
P = desired probability that desired gene is in library
N = required number of colonies
When the size of the whole genome from which the gene was extracted, such as with humans, for making a library of all the genes, how do you narrow down the selection?
By using the transcriptome only.
Transcribe all the coding DNA into RNA with an RNA polymerase
Use Reverse transcriptase (like from retroviruses) to convert transcriptome back into DNA for ligation.
Reverse transcriptase requires primer, use poly(T) primer complementary to poly(A) tail of mRNA!!
(from this RNA/DNA complex degrade RNA with alkali) (first DNA strand loops back on itself and self-primes)
What problems arise when Reverse transcribing cDNA from mRNA transcriptome? (with second strand synthesis) And how to overcome this?
When degrading remaining RNA strand on DNA/RNA mix, either by alkali degradation or by RNA nucleases.
A little bit of sequence information will be lost, (either because of the 3’ end loop of DNA after alkali degradation or the remaining piece of RNA primer after RNA nuclease digestion, (both of which have to be cut off))
So add a poly(C) homopolymer extension to 3’ end of first cDNA strand (using terminal transferase enzyme) and then prime for second DNA strand polymerisation using complementary Poly(G) primer!
How to enrich for (improve odds for getting) your desired gene? (when creating gene library, reducing necessary size of library/no of clones required)
Use only the chromosomes its on (determined by mapping perhaps)
Use mRNA from tissues specifically in which your gene is expressed! (to reverse transcript to cDNA)
Environmental factors known to cause expression of your gene (then use resulting enriched transcriptome)
Size fractionation (filtering out fragments larger or smaller than your gene is expected/known to be)
plus others.
How to identify our desired gene from gene library (of clonal colonies)?
Test obvious phenotypes (like making an enzyme) although this requires the gene to be expressed!
Transfer clonal colonies to membrane, lyse DNA, hybridise with labelled probe for desired gene.
(if sequence of gene is unknown then can use protein sequence, although different codons for same sequence possible so variety of probes needed, minimised by knowing which codons more commonly used by organism, and perfect match not required)
Purpose of histidine tagging in expression vectors?
Allows purification of His tagged protein! by exposure to Nickel ion containing medium which aggregates histidine residues, can then wash off non-histidine tagged proteins.
Components of pET system?
pET phagemid, contains T7 promoter that only binds T7 RNA polymerase.
Bacterial Chromosome contains T7 RNA polymerase gene, under the control of the lac operon, so when cell exposed to lactose (or IPTG) it triggers lac operon and so T7 RNA polymerase expression, and subsequently gene of interest expressed (under control of T7 promoter.)
How to incorporate genes into plants?
Ligate gene into Agrobacterium Ti (tumour inducing) plasmids. These pathogens transfer their genes, or our transgene into the DNA.
PCR temperature cycles?
2 primers, flanking site of interest.
Melt dsDNA strands apart. 94deg
Anneal DNA primers correctly by slowly cooling. 40-60
Extend (at optimum temperature for [taq] polymerase approx 72deg)
Repeat (because first products too long!)
What are T-vectors? how to ligate DNA products of Taq polymerase PCR?
Taq polymerase for adds extra adenines (A) on the 3’ hydroxyl ends of its products.
T-vectors are plasmid vectors with single 5’ overhanging Ts that pair to the As TOPO also acts as ligase (in place of T4 DNA ligase)
What is reverse transcription (RT) PCR?
Amplifying mRNA by including a reverse transcription step (perhaps catalysed by a multifunctional DNA polymerase like Tth)
1) PolyT primers for mRNA polyA tail for first DNA strand
2) To prime for second DNA strand formation first use terminal transferase to add poly(G) to 5’end and prime with PolyC primers!
Quantitative/real time PCR technique?
Used to assay level of target DNA/mRNA in sample.
Level of product indicated in real-time (after each PCR cycle) by 3 different fluorescent probe techniques.
1) Light-cycler assay with dsDNA binding cybergreen dye
2) Taqman assay
3) Molecular beacon self-quenching probes
Critical to use control (of known conc. mRNA) to allow absolute quantities to be determined
What is a Taqman assay technique in real-time (quantitative) PCR?
Add a self-quenching probe specific to the sequence to be amplified.
Taq removes and degrades the probe as it finds it during extension phase of PCR. Degraded probe can no longer self quench and so fluoresces
So fluorescence level proportional to amount of sequence,( to probe binding and subsequent degradation.)
How does PCR mutagenesis (Quickchange) work?
Amplify plasmid with mutant primers. Digest original/template DNA using a restriction enzyme specific for methylated DNA only. (e.g. Dpn1)
Transform E.coli with the ‘gapped’ plasmids and E.coli will ligate the ends.
Methods of ‘random’ mutagenesis?
Site-directed mutagenesis with a degenerate (random amino acid generating) sequence primer
Error prone PCR introduces random mutations
Recombination based technique:
DNA shuffling: produces hybrid of homologous genes/alleles.
What is surface display of proteins? and how is it useful in identify improved mutant proteins following random mutagenesis? (site directed or DNA shuffling or error prone PCR)
System where proteins are displayed on surface of host bacterium, yeast or bacteriophage.
Can then test substrate binding properties of random mixture of displayed mutant proteins on fixed substrates.
Allow phages or bacteria to bind, and wash off weaker binding mutants. (varying temperature etc)
As the remaining mutants carry their DNA with them you can analyse/or amplify and remutate it
What is chemical compartmentalisation and droplet assay?
(used after random mutagenesis to assay multiple mutants)
Trap individual mutant/recombinant plasmids in water droplets in an oil-water emulsion.
In each droplet is machinery to transcribe and translate plasmid!
Separate drops into different wells and assay function of protein.
Or FACS fluorescent activated cell sorters can be used if mutant is an enzyme that can be caused to maked fluorescently labelled product.
