Gene Cloning and Manipulation Flashcards
Typical PCR programme
1) Heat to 94°C for 3min to denature. For subsequent cycles 1min.
2) Lower to 40-72°C (avg 55°C) for annealing (1min).
3) Raise to 72°C for extension (1min).
4) Repeat 25-30 times.
5) Cool to 4°C.
PCR reaction mix
Primers (excess)
dNTPs
Template
Heat stable polymerase
Mg2+
What determines annealing temperature?
5°C lower than melting temperature of primers - determined by GC content and length of primers.
How do the quantities of products increase?
Geometric increase in strands with primer (or complement) at both ends.
Arithmetic increase in strands with primer at one end.
What are the properties of Taq polymerase?
94kDa
5’-3’ DNA polymerase
Incorporates at 50-60nt/sec at 72°C
5’-3’ exonuclease
No 3’-5’ proofreading
Half life at 95°C of 40min (so significant loss of activity during full set of cycles)
How are products identified?
Run on agarose gel.
Stain with ethidium bromide - intercalates into DNA helix and fluoresces.
Cut out bands and digest with enzymes to confirm identity.
Why is PCR useful?
Can have very specific amplification
Readily automated
Works with partially degraded DNA
Works with very tiny quantities of DNA
Where may sequence information come from before doing PCR?
Might already know sequence - part of a larger fragment previously sequenced or from published genome.
Might know amino acid sequence encoded - use degenerate primers.
DNA sequence may be part of a family with conserved features - design primers for those features.
Why use degenerate primers?
If only the amino acid sequence is known, degeneracy of amino acid code means DNA sequence can’t be exactly known. Degenerate primers are created to cover all possible coding sequences and used as a mixture.
Sometimes it is possible to guess the most likely sequence by looking at preference in other genes.
What compromise must be made in primer design?
Between a short sequence that can be guessed accurately but will be more non-specific and a longer sequence with degenerate guesses but more specific binding.
Other factors in primer design
Length: ideally 20-30bp.
Must match at 3’ end for efficient extension.
C or G at 3’ end aids efficient extension.
Shouldn’t be able to fold back on themselves.
Primer pairs should have similar annealing temperature to the template DNA.
Primers dimers should be avoided.
PCR application: Thylacine
Extinct Australian marsupial wolf.
Not known if native to Australia or survivor of ancient South American marsupial wolves.
Amplification of mitochondrial DNA from skin and museum specimens (e.g. from 200yo mummified Thylacine).
Conclusion: convergent evolution.
PCR application: Neanderthals
DNA extracted from 3 individuals.
4 billion nucleotides in fragments of <250bp amplified and sequenced.
Compared to modern humans around the world.
Showed breeding with them took place, before divergence of Eurasian groups.
Neanderthal variants not found in Sub-Saharan populations.
Polymerase errors
If error occurs in an early cycle, will be found in a lot of product molecules.
Taq has error rate of 1 in 10^4.
Polymerases with 3’-5’ proof reading activity reduce error but are slower and more expensive.
Examples: Vent, Pfu, Pwo.
Pfu Ultra is 18x better than Taq.
What product size is PCR most efficient for?
Up to 2-3kb.
A mixture of polymerases can sometimes be used to amplify larger pieces.
Longer fragments also require increased synthesis time in cycle.
What causes non-specific priming?
May be other places in genome with sequence similar enough to target that primers anneal.
Degenerate primers can increase the problem as there is a mixture of sequences.
How can non-specific priming be reduced?
Change the annealing temperature.
Increasing the concentration of Mg2+ reduces the fidelity of Taq.
Adding DMSO inhibits DNA 2° structure.
Alter PCR technique.
What are alternative PCR techniques?
Touch-down PCR.
Hot start PCR.
Nested PCR.
What is touch-down PCR?
Start with annealing temperatures higher than predicted and reduce each cycle.
First successful reactions occur under most stringent conditions.
What is hot start PCR?
Polymerase/Mg2+ not added until denaturation temperature reached.
Both can be added manually.
Alternatively polymerase incorporated into wax beads that don’t melt until denaturation temperature can be included in initial mixture.
What is nested PCR?
Two rounds of PCR.
Second uses products of first but with new primers designed to anneal within desired product.
Highly unlikely to get mis-priming in second reaction.
What is inverse PCR?
Use if know the sequence of one portion of the fragment, and not of the ends.
1) Cut DNA with enzyme that doesn’t cut within known sequence
2) Ligate fragments under conditions favouring self-ligation
3) Cut with enzyme that cuts within known sequence
4) Target DNA now has known sequence at both ends and can be used in PCR
What is RT-PCR?
Reverse transcriptase PCR.
Used to amplify RNA sequences as DNA copies.
Equivalent to cDNA cloning.
Normal PCR preceded by replication reaction at normal temperatures with reverse transcriptase.
What is asymmetric PCR?
Use an excess of only one primer.
When lower concentration primer has been exhausted, other primer allows linear accumulation of single strand DNA.
Used to be useful for sequencing preparation, but now ssDNA not needed for this.
What is qPCR?
Quantitative PCR.
Allows tracking of build up of product as reaction progresses. More initial template = more product.
Run products of different number of cycles on agarose gel. More template = brighter band.
Only works in limited number of cycles, when increase is linear. Saturation occurs after too many PCR cycles.
How can product accumulation be detected in real time?
Include a dye that binds to dsDNA and fluoresces.
Include a specialised oligonucleotide probe hybridising to a region within the target sequence.
Details of qPCR with fluorescence.
Common dye: SYBR green.
Intensity of SYBR green fluorescence above background measured (CT) and used to quantitate the amount of newly generated dsDNA.
SYBR green binds to any dsDNA so isn’t specific to correct product.
Details of qPCR with oligonucleotide
Probe labelled at 5’ end with fluorescent reporter and at 3’ end with a quencher.
When reporter and quencher are close, i.e. on same molecule, no fluorescence.
5’-3’ exo activity of polymerase cleaves 5’ label from probe, allowing fluorescence.
Fluorescence quantified, so quantity of product quantified.
What is conventional cloning?
Indefinite propagation of DNA sequence using an organism to carry out replication.
What is the standard cloning vector?
The pUC vectors and their derivatives.
What are the features of pUC vectors?
bla gene: resistance to ampicillin, encodes a beta-lactamase
lacZ’ gene: encodes first 146aa of beta-galactosidase with host genome encoding remainder, except residues 11-41 (M15 deletion). ß-galactosidase breaks down artificial substrate X-gal to produce blue pigment
lacI gene: encodes Lac repressor
(MCS) multiple cloning site: a collection of recognition sites for restriction enzymes, found in lacZ’ gene
Cloning procedure
1) Extract DNA from organisms in question
2) Digest DNA to be cloned with appropriate enzyme (e.g. BamHI)
3) Digest vector with same enzyme
4) Mix and ligate - generates three types of molecule
5) Transform E. coli with ligation products (heat shock/electroporation/CaCl2)
6) Plate on medium containing ampicillin, IPTG (inducer of lacZ - inactivates the repressor) and X-gal
How are correctly transformed cells detected?
Cells which took up nothing or only the insert are killed by ampicillin.
Cells which took up plasmid with no insert survive but have active lacZ’ gene, so are blue becuase of X-gal.
Cells which took up plasmid with insert will survive and have inactive lacZ’ gene, so are white.
How can self-ligation of the vector be prevented?
Treatment with alkaline phosphatase removes terminal phosphate groups on the molecule. Only hydroxyl groups are left. These cannot be self-ligated by DNA ligase.
The insert DNA still has terminal phosphate groups and so can be ligated into the vector.
Resultant nicks at each vector-insert junction are repaired within E.coli cell.
What is EOP?
Efficiency of plating.
Measure of how well phage infect a bacterial cell.
What is the difference between E. coli C and E.coli K?
E. coli K has a restriction enzyme that degrades incoming phage DNA and a methylating enzyme to protect its own DNA.
E. coli C has neither.
How do the differences between E. coli C and E. coli K affect EOP?
Phage grown on E. coli C can re-infect strain C with EOP = 1
Phage grown on E. coli C have low EOP on E.coli K, EOP = 10-4
Those that survive on E. coli K can re-infect strains C and K with EOP = 1
Phage that grow on C have unmethylated DNA so are unprotected from restriction enzymes when trying to infect K.
One in a few thousand gets methylated before degredation so can then replicate in K.
Features of Type I endonucleases
Require ATP to function.
Methylase and restriction enzymes are subunits in same complex.
Cut a random distance (at least 1000bp) from recognition site.
Features of Type II endonucleases
Require Mg2+ to function.
Restriction and methylation enzymes are separate in Type II and Type IIS.
Most recognise and cleave DNA at same site.
Type IIS cleave outside of recognition sequence, on one side.
Type IIG cleave outside of recognition sequence, on one side, if sequence is continuous. If discontinuous, the cleave at either end, releasing a fragment containing the recognition sequence.
Features of Type III endonucleases
Require ATP to function.
Methylase and restriction enzymes are subunits of the complex.
Recognise two separate, non-palindromic sequences that are inversely orientated.
Cut 20-30bp after the recognition site.
What do sticky ends allow?
DNA fragments can only be joined together if sticky ends are complementary.
Different enzymes can give the same sticky end.
Ends generated by different enzymes can be complementary but may not re-generate a restriction site.
Cloning with sticky ends can be directional if enzymes produce different ends.
What do blunt ends allow?
Can be joined regardless of sequence.
Not as efficient as sticky ends.
Sequences cut to give sticky ends can be ‘filled in’ to produce blunt ends.
How can PCR be used to create sticky ends?
Taq polymerase has a terminal transferase activity, so adds an A residue to the 3’ end of PCR products.
Vector that gives a single T-overhang (e.g. pGEM-T easy) can be directly used for cloning PCR products.
Some polymerases do not give overhangs, e.g. Pfu. PCR products can be cloned in with blunt ends, but less efficient and non-directional.
How can PCR be used to add restriction sites?
Incorporate restriction sites into the primers.
May be necessary to add extra nucleotides to the end of the primer, to give the enzyme something to bind to as well as the recognition site.
Can then be ligated into vector. Sequence to check correct PCR.
What must a vector have?
Origin of replication
One or more markers (usually antibiotic resistance)
Suitable restriction sites
What properties are often needed in a vector?
Easily handled (not too large)
Markers for DNA insertion, e.g. lacZ’
High copy number. pBR322 has low copy number, pUC have high. Same replication origin, so probably mutation in regulation system.
Debilitated
What characterisitcs are desirable in a host?
High efficiency of transformation. Depends on ability to take up DNA and lack of enzymes to degrade it once taken up.
Recombination deficiency to maintain plasmid stability.
Debilitation.
Other markers.
What is endAI?
Endonuclease deficiency, makes it easier to prepare intact DNA.
What is hsdR17?
Inactivates host restriction system.
What is supE44?
Chain terminator supressor mutation.
Strain carrying this can be used as a host for viruses which have been debilitated by incorporation of a chain termination mutation.
What is thi-I?
Mutation in a gene for one of the enzymes of thiamine biosynthesis.
Gives some biological containment.
What is recAI?
Reduces recombination activity.
What is gyrA?
Inactivates a DNA gyrase.
Reduces recombination levels further.
What is relAI?
To do with control of RNA synthesis.
What is mcrA?
Inactivates a host endonuclease.
What is Δ( lacZYA-proAB)?
Deletion extending from lac operon to the proAB genes involved in proline biosynthesis.
What is F’(traD36)?
Contained on an F’ plasmid.
Mutation in a gene that is required for the plasmid to transfer itself to other cells.
What is F’(proAB+)?
Found on F’ plasmid.
Genes for proline biosynthesis that were lost from main chromosome.
If host is grown in absence of proline, the F’ plasmid is retained.
What is F’(lacI9)?
Gives high levels of the lac repressor.
Allows better control of the lacZ gene.
What is F’(lacZ∆M15)?
Rest of the *lacZ *gene that isn’t in the vector.
What are the advantages of bacteriophage lambda over plasmids?
Easier to handle large numbers.
Can be more efficient.
Can have larger inserts.
What can happen to lambda after it infects a cell?
Can replicate directly, producing more phage and lysing the cell.
Can insert its DNA into the host’s, forming a lysogen. It lies dormant until activated and then replicates lytically.
What are the features of the lambda genome?
~48.5kbp long
Linear but can circularise in host cytoplasm due to 12bp overhang at 5’ ends (cos sites).
What types of lambda vector are there?
Insertional vectors: extra DNA is inserted into the lambda genome.
Replacement vectors: non-essential genes are removed by digestion and replaced by insert cut with same enzyme.
Phage can take 37-52kbp of sequence, inserts are usuall ~ 25kbp.
How are recombinant genomes packaged?
Can be packaged in vitro, using extracts from induced lysogens.
Requires proteins and cos recognition sites on DNA to be packaged.
What is a cosmid?
Plasmid containing the lambda packaging site, cos, and a selectable marker.
Can be packaged in vitro if insert is large enough and introduced into cells by infection.
Once in cell, cosmid replicates like a normal plasmid.
What is the advantage of a cosmid?
Only a few kbp, so rest of the ~50kbp of sequence required for packaging to occur must be made up from the DNA to be cloned.
So packaging provides a selection for the cloning of large stretches of DNA.
What are the disadvantages of using cosmids?
Bits of the cosmid can be easily deleted after infection.
Can have problems with recombination.
What is a BAC?
Bacterial Artificial Chromosome
Modified F plasmid.
Can take inserts up to ~300kbp.
Usually introduced into E. coli cells by electroporation.
What is found in a BAC?
Recognition site for restriction enzyme. (e.g. HindIII)
Selectable marker (e.g. ChloramphenicolR).
Origin of replication (e.g. oriS)
Replication promoter (e.g. repE)
Genes to maintain low copy number (e.g. parA and parB)
May have cos site to allow packaging into phage, but requires size constraint.
Why ensure low copy number?
Reduces the chance of recombination, and therefore rearrangement/deletion.
Deletions are more likely to occur when cloning large pieces so need to compomise on yield to favor stability.
What is a PAC?
Phage artificial chromosome.
Based on bacteriophage PI.
Accept similar size of insert to BACs.
Normally introduced into host cell by electroporation.
Can be packaged in vitro and host cells infected, but imposes size constraints.
