Gene cloning and manipulation

Taq polymerase

• 72-75C optimum

• No proofreading

Requirements of primers

• 20-30bp for specific priming

• C/G clamp to prevent drop off

• Roughly same temperature for annealing step

• Avoid complementarity to prevent primer dimers

• Avoid secondary structure (e.g. hairpin loop) formation

Methods of improving primer specificity

• Decrease concentration Mg2+ to decrease mispriming (Mg2+ offsets -ve charge on phosphate backbones, reducing repulsion)

• Touch down PCR

• Hot start PCR

• Nested PCR

Describe touch-down PCR and draw a graph

• Start with highest temperatures, where all will be bound

• Decreasing temperature every cycle

• First successful reactions will be under the strictest conditions

Describe hot-start PCR

• Polymerase is made to be completely inactive until annealing temperature is met

  • Can either add polymerase manually when temperature is reached, or use JumpStart Taq which is inactive below 70C

Describe nested PCR and draw a diagram

• In round 1, primers are sometimes non-specific and can bind in multiple places

• In round 2, a nested primer is used that will bind inside the target sequence, but not inside the misprimed sequence

• Used when round 2 primer could not be used in round 1 as too non-specific, but once sequence is narrowed down, it can be used

What is the multiple cloning site

• Series of restriction sequences that will be recognised by various restriction enzymes

Where is the MCS in pBluescript and why is this useful

• Within the functioning LacZ’ gene

  • DNA insertion will inactivate the gene, allowing for blue(non-insert)/white(insert) selection

What is the LacZ’ gene?

• Encodes beta-galactosidase

  • When combined with the rest of an E.coli genome, will cause colonies to appear blue when exposed to IPTG and X-gal

T7 phage promoter

• Increases replication rate

Purpose of ampicillin resistance gene in a plasmid

• Can kill all cells that didn’t take up the plasmid using ampicillin

Histags

• Helps purifiy proteins

  • Follow with thrombin site to cleave tag once purifiedW

What are good characteristics of cloning plasmids

• MCS

• LacZ’

• Ori site

• T7 phage promoter

• Ampicillin resistance

• Histag

  • Thrombin site

OriC site

• Promotes plasmid replication inside the cell

What is favourable when choosing an E.coli strain to transform into

• High efficiency of transformation

• Lack of DNA-degrading enzymes

• Recombination deficiency to maintain plasmid stability

Blunt cloning

• PCR usually makes blunt ends

• Treat plasmid with blunt-end RE (e.g. EcoRV)

• DNA ligase

  • Non-directional

TA cloning

• Taq adds a non-templated A to the end of PCR products

• Clone using a different high-fidelity polymerase

• Use taq for final cycle, will add an A

• Clone into commercially available vector that has a single T overhang

• Efficient

• Non-directional

Gibson’s assembly, draw a diagram

• Joins multiple fragments directionally, regardless of sequence

• Use in-silico primer design to create a series of fragments with overlapping ends

• Treat with T5 exonuclease that causes 3’ overhanging sticky ends

  • Annealing with polymerase will seal the nicks

Golden gate cloning

• Simultaneous and directional assembly of multiple DNA fragments using type IIS restriciton enzymes and T4 DNA ligase

  • The RE cleaves DNA just after recognition sequences so recognition sites are removed from the insert

RT/qPCR

• Before first PCR replication, add reverse transcriptase at a normal temperature

• Will turn all mRNA into DNA

  • Amount of DNA produced during a known number of PCR cycles correlated with the amount of mRNA started with

SYBR green dye and qPCR

• Used in qPCR

• Binds to dsDNA and fluoresces

• Intensity of fluorescence above background level is measured

• Number of PCR cycles to generate fluorescence above a threshold value is the CT

Sequence-specific fluorescent probes and qPCR

• Fluorophore at 5’ end, quencher at 3’ end

• Probe binds to DNA downstream of primer, no fluorecence

• 5’ → 3’ exonuclease activity of DNA pol will cleave and release the fluorophore

  • Fluorescence is now detectable

Purification of his-tagged proteins

• Immobilize on columns lined with nickel ions

• Wash away unbound

• If cleavage site present: add protease to release protein from column

  • If lacking cleavage site: elute by imidazole that competes for cation sites and displaces fusion protein

Considerations when expressing proteins in E.Coli

• Doesn’t use splicing, so unless cDNA is used, introns will be expressed

• Protein may not be easy to isolate without loss of biological activity

• Protein may be lysed or secreted

• Could be toxic

Describe immunoprecipitation as a way of identifying interacting proteins

• Antibody fixed to protein A/G beads that binds specifically to protein of interest

• Verify that antibody only recognises that protein and none others in that mixture

• Magnets can capture protein A/G beads, bringing out the protein of interest and also any proteins that are attached to it

  • Mass spec to determine interacting proteins

Describe the yeast two-hybrid system for identifying interacting proteins, draw a diagram

• S.cerevisiae transcription factor GAL4 has a DNA binding domain and an activation domain. When joined together, activate transcription

• Joining doesn’t have to be direct - can be activated by bridging with proteins

• Synthesize DNA binding domain to protein of interest, and activation domain to a variety of query interaction proteins

• See which combinations cause transcription

Mutagenesis PCR, draw a diagram

• Mutant olignoucleotides that are entirely complimentary except for the desired point mutation

• Transform into E.coli

• Will either replicate (introducing the point mutation on both strands) or repair

Simply, what are three ways of reducing protein expression?

• RNA interference

• CRISPR/Cas9

  • Gene inactivation by homologous recombination

Reducing protein expression with RNA interference

• Post transcriptional reduction

• Long dsRNA processed into siRNA by RNAase III (Dicer)

• Made of one guide strand and one passenger strand (that will be destroyed)

• siRNAs assemble with Ago2 to form RISC

• Perfect complementarity

• Duplex formation causes endonucleolytic cleavage by Ago2

• Remaining mRNA degraded in general cellular decay pathways

What determines which strand is the guide and which is the passenger in siRNAs

• The stability of each 5’ end

When can and can’t siRNA be used in humans

• Can’t when need to introduce long dsRNA as triggers an interferon response (mistaking for virus e.g. rotavirus)

• Use synthetic shRNA to transfect, which will then be processed like miRNAs to give rise to the guide strand that still targets mRNA without an interferon response

Describe the components of CRISPR/Cas9 used in gene editing

• Cas9 dsDNA nuclease

• gRNA that combines crRNA and tracrRNA (specific to the sequence being edited, generated by cloning of oligonucleotides specific to the target DNA)

How does CRISPR/Cas9 edit genes

• gRNA binds with perfect complementarity

• Cas9 causes ds break that triggers repair mechanisms

• Repairing is good but not great, so can make error

• If repair is perfect, gRNA binds again and Cas9 cuts

  • Repeats until microinsertion or deletion is complete

What do big deletions using CRISPR/Cas9 editing require?

• Two gRNAs for two ds breaks that ligate together to completely remove that section of DNA

How can dead Cas9 be used in gene editing?

• Blocks transcription

• Can be bound to other enzymes or fluorophores

  • Method of specific recruitment on the genome

Gene inactivation by homologous recombination

• Knock out of endogenous genes by swapping them with a mutant gene

• Insert a selectable marker into a clone of the gene desired to be disrupted

• Introduce into non-replicating DNA

• Select for maker, only way for cell to survive is for it to replace the existing gene in the genome with the disrupted marker gene

• Endogenous gene is now replaced by disrupted copy

Analysing protein function using microinjection of transgenic animals

• Microinject DNA into pronucleus of fertilized eggs

• Random integration

• Transplant to psuedopregnant mother

  • 10-20% offspring will develop into the transgenic organism

Analyzing protein function using cultured embryonic stem cells and transgenic animals

• Undifferentiated cells can be manipulated

• Reinjected into developing embryo

• Identify suitable cell lines that have integrated the DNA using PCR

• Develop

• Form a mosaic animal

  • Can be mated to WT to achieve heterozygous mutant and if mutation is dominant, will be completely presenting the mutation

Use of bromophenol blue in gels

• Visualise loading

  • Can monitor how long the gel has been running

Advantage of using two different restriction enzymes to clone a DNA fragment into a plasmid

• Directional

How to check insert orientation without sequencing, draw a diagram

• Restriction digest using two enzymes that cut asymmetrically within the insert

  • Analyse using agarose electrophoresis alongside molecular weight markers

Reasons why PCR has failed, besides forgetting to add any components

• DNA template quality (degraded or contaminated)

• Primers have sequences that form primer dimers or secondary structures e.g. hairpins

• Incorrect annealing temperature

• Too little Mg2+, too much primer-template repulsion

Why could a variety of sizes of DNA bands be obtained after a PCR when only one is expected? How to resolve?

• Non-specific primer binding

• Increase annealing temperature

• Decrease Mg2+ concentration

  • Change primers

How much DNA is required for LCN PCR?

• 100-200pg of cells - roughly one billionth the mass of a grain of rice

What are advantages of using a reporter gene in an assay for expression

• Analyse promoter function

• Can easy be visualised (GFP) / assayed (beta-galactosidase) to interpret expression from a particular promoter

• Analyse other regulatory elements or sequences (enhancers, miRNA binding sites)

• Can compare promoter function

• Study localisation

Describe how DNA footprinting is carried out

• DNA is one end-labelled using 32P

• Protein incubated with dsDNA

• Mildly digested with DNAase, each DNA molecule is cut once

• Separate on agarose

• Visualise radioactivity

• DNA without protein will have a continuous fragment ladder (cuts at a random points on every strand)

  • DNA and protein will have a gap (footprint) where DNA has been protected and remains associated with protein

Where are hydrophobic amino acid interactions most important in a protein?

• Core

• Stabilize structure

What are charged side chains of amino acids especially good at doing?

• Stabilizing helix dipole of alpha helices