Gene cloning and Manipulation Flashcards
(24 cards)
PCR general workflow
Add primers, template, nucleotides, polymerase (taq-thermostable) to cycler.
1st phase: melt dsDNA template at 95C. 2nd phase: anneal primers at 55C. 3rd phase: polymerise at 72C. Never 100% efficient so in practice doesn’t quite double every cycle.
Primer design
Using the sequence of the template (if have sequenced or can be found on NCBI) or using sequences of analogous/similar genes found with BLAST/horizon/other databases, or by reverse engineering from the protein sequence (degeneracy means you have to compromise between short accurate vs long inaccurate seq). cDNAs can be bought commercially.
Consider: both primers have same annealing temp; 20-30bp (but can be more); C/G at 3’ end best for extension; avoid mismatches at 3’ end (clamp primer on); avoid internal or primer-primer complementation. Incorporate the restriction site/sticky end @5’+ flanking nts => digestion efficiency (#depends on enzyme)
PCR troubleshooting
Many contaminants amplified from original sample/mispriming: use longer primers with higher CG content, increase annealing temperature. “Hot start”- only add taq after primers fully annealed i.e. in 3rd phase. “Touch down”- start with much higher annealing temperature for a few cycles, then decrease to ensure high concentration of desired product. Decrease concentration of DNA duplex-stabilising ions (Mg2+). “Nested PCR”- 2 consecutive rounds with different primer pairs.
High polymerase error rate: High fidelity polymerases with lower error rates e.g., containing 3’-5’ exonuclease action to “spellcheck” new lagging strand (taq only has 5’-3’ exonuc activity).
Truncated products (5kb up): extend polymerisation time; use mix of polymerases to decrease falloff rate (e.g., Taq and Phusion); reduce denaturation time and increase pH to reduce depurination (destabilises DNA @ high temp, low pH. Use a commercial kit.
Genomic contaminant in cDNA: use exon flanking in primer design. Digest DNA with enzymes then inhibit before reverse transcription to avoid cDNA digestion.
Vector components (e.g., pBluescript II SK)
MCS (multiple cloning site): recognition sites for restriction enzymes
lacZ’ gene: fragment of gene that encodes B-galactosidase (the rest in the host)- together produce functional B-galactosidase, break down X-gal, produce blue pigment. MCS disrupts lacZ’ in original plasmid, so if fragment cloned in, gene disturbed, lacZ’ non-functional+ no X-gal breakdown= white colony. If insert unsuccessful, lacZ’ functional, blue colony. White colonies= ones with plasmid that has the insert (i.e., the one you want).
Antibiotic resistance gene, e.g., chloramphenicol, ampicillin: bla gene product breaks down ampicillin- colonies surviving on ampicillin plate have a plasmid (transformed).
Cloning a fragment into a vector (workflow)
Add PCR’d fragments to linear vector, mix and ligate, transform into E-coli hosts, select white colonies growing on ampicillin+X-gal+IPTG (lacZ inducer), if vector as above.
Why E-coli? High efficiency on transformation (especially after CaCl2+heat shock)+ unlikely to reject plasmid; plasmid stability maintained by low recombination levels.
Can reduce background (self) ligation by treating with alkaline phosphatase- remove terminal phosphate group, OH groups at the end can’t self ligate, but ligase can ligate it onto insert (not treated with phosphatase)- nick at insert-vector junction fixed by host.
Common methods of cloning into a vector (excluding Gibson’s and Golden gate)
Blunt: non-directional, not as efficient as “sticky”.
TA: Taq adds non-templated A to 3’ end (terminal transferase activity)- even when high-fidelity pol used initially to amplify+ make blunt product. Clone into vector with T overhang- more efficient, still non-directional.
Sticky end/type II restriction enzymes: use 2 types to make cloning directional. (NB methylation protects DNA from restriction degradation). Type I+III don’t cut in recognition seq so II most useful.
NB Isischizomers: create same sticky ends but if fragment joined, neither recognition site created.
Gibson’s assembly method cloning workflow
Gibson’s assembly method: almost any fragments, can join >2 overlapping fragments at once in single isothermal reaction. No specific restriction site needed but need overlapping ends. No scar, directional, efficient. Design primers for product with correct overlaps to order fragment assembly; add to assembly reaction with T5 exonuclease ( create ssDNA 3’ overhangs, facilitate complementary annealing), polymerase (fill gaps within annealed fragments), ligase.
Golden Gate cloning
Golden gate: 2+ fragments into 1 piece, simultaneous and directional. Use type IIS restriction enzyme to cleave outside recognition seq (which is distal to cleavage site)- removes recognition seq from assembly. Destination vector has BsaI recognition seqs in opposite directions, creating 4 base overhang. Primers have complementary overhangs, allowing insertion w/ ligase.
Extra considerations when cloning
Extra considerations: When picking restriction sites for a vector and fragment, consider if the recognition site is close to where you want your insert and if it is similar to a sequence in your fragment (may cut fragment to be cloned into vector instead ☹). If cloning a fusion protein, the number of extra nucleotides in your primer may also need to be adjusted and the stop codon either added or removed depending on whether you want to clone the tag onto the N or C terminal of the original fragment.
It is good practice to check that the final clone is correct by sequencing.
Non-PCR cloning
Sub-clone from existing clone if restriction sites in convenient locations, especially useful when MCS conserved between clone.
Synthesise the gene/ORF you want (easy but expensive)- especially useful when no clones available/ expression increased by optimising sequence.
Introduce point mutations/reshuffle/insert/delete/add fusion tags etc into existing sequence- available through e.g., GenScript
Analysis of gene expression using PCR
RT- replication at normal temp with reverse transcriptase, then normal PCR. Reverse transcriptase can be from AMV or MMLV, usually optimised for better yields e.g., Superscript IV based on MMLV: Reduced RNase H activity so greater first strand cDNA yield, products up to 12kb, fully active at 42C, can use either total or poly(A)+RNA. Need primer with free 3’OH.
If want to synthesize other strand (i.e., just cDNA), can use RNase method: RNase targets DNA:RNA hybrid, cuts RNA only.
qPCR
* SYBR Green- fluoresces when binds dsDNA- intensity above background measured- # cycles to get intensity above threshold(above background, start of exponential curve increase)= Ct
* Specific probe/”taq-man”- seq specific probe hybridises part of target seq. 5’ fluorophore, 3’quencher- when both close, no fluorescence. When probe anneals target DNA, 5’-3’ exonuclease removes 5’ end of probe->fluorescence.
Note that the fold-difference of a transcript of interest upon treatment be different between a qRT-PCR and a reporter assay: if transfected genes vs endogenous genes being measured, may respond to a promoter differently. Different lengths of reaction and measuring different molecule types (RNA vs. protein)
In vitro assays and transcription
In vitro assays
Binding, footprinting, UV crosslinking, editing assays, assays for structural analyses.
In vitro transcription
Plasmids with phage promoters recognised by specific phage polymerases, synthesise RNA in vitro, e.g., T3 and T7 promoters in pBluescript. Phage direct synthesis of own RNA polymerases in host, specific for phage promoters.
Sequence clones into vector downstream from promoter (MCS is between 2 promoters)- orientation and choice of polymerase determines strand transcribed; plasmid linearised at end of seq to be transcribed; incubated with phage polymerase in vitro; pure transcript downstream of phage promoter produced; transcript can be capped (5’)/polyG included in template.
Recombinant proteins expressed w/ promoter, ribosome binding site, start codon, transcription terminator.
Common E-coli promoters:
* Pl (from lambda- functional at 30C, not at 42C);
* PlacZ- inactivated by IPTG, ctrled by lac repressor;
* Ptac+Ptrc- synthetic, -35 of trp operon+ -19 of lac, ctrled like PlacZ; ParaBAD- araBAD promoter, activated by arabinose (which binds AraC, dimerises, promotes RNA Pol binding);
* T7- IPTG induces lac promoter->promotes T7 RNA Pol->transcribes T7 promoter-> promotes required gene.
e.g., pET vector system- host base on Ecoli BL21, lysogenic for modified Lambda (carries T7 Pol under lac promoter ctrl)
In vitro basal transcription assays find quantity and accuracy of initiation:
Incubate DNA template w/NTPs+ nuclear extract (low salt to extract histones) or purified Pol II and other factors, hybridise purified RNA with 5’ labelled DNA oligo, incubate with dNTPs and reverse transcriptase, analyse labelled DNA with denaturing gel.
Fusion proteins creation workflow
Fuse protein seq for tag to N-terminus of gene for protein of interest (can remove later if use vector allowing protease cleavage at protein-tag junction, then separate w/ chromatography); His tag (6xHis residues, purification. NB proteins with longer His runs/ binding Nickel can co-elute); Epitope tag (label- good for immunoprecipitations, immunofluorescence, immunoblots etc.); MBP (solubility); GFP (localisation); GST (pull-downs- immobilise on column and look for interactions w/ other proteins); X-tag (in vitro assays); phage display (coat protein fused on, displayed on surface- good for IDing interacting ligands).
Proteases from blood clotting cascade: Factor X activated-> prothrombonin converted to thrombonin->fibrinogen converted to fibrin.
Vector for fusion protein needs appropriate translation signals, in order going downstream: promoter, ribosome binding site, ATG, extra bit of protein code (tag, ends up on N terminus, i.e., the start), ORF.
Fusion proteins for purification (hint: tags)
Expression induced in E-coli, then crude lysate prepared. Purified on columns according to tag. Can all be eluted with protease or respective competitors:
His: Sticks mobilised nickel ions, eluted with imidazole to get whole fusion protein
GST: Glutathione sapharose column, elute with glutathione. Often cleave off as GST large
MBP: increases solubility. Amylose column, eluted with maltose (binds MBP, not column as His/GST)
Proteins can be further purified by repeating column purification with a different tag (if 2+ tags present on fusion protein), by gel filtration, immunoprecipitation. The tag could also be cut off and the untagged protein run through again to get just the original protein.
Considerations when making fusion proteins
No introns or use cDNA- no splicing in E-coli. Translation shouldn’t use rare tRNAs/ these should be provided. Protein may aggregate/form inclusion bodies if misfolded (easy to isolate, hard to solubilise, may lose activity- inclusion body formation depends on redox state- can adjust)
Protein may be proteolyzed- Ion- cells don’t have gene commonly responsible for proteolysis, but can lead to mucoidy and long filamentous cell formation (inviable). ompT product can also lyse protein.
Protein secreted- helps purification/avoid proteolysis- fusion with secretion protein/signal e.g., MalE causes export to periplasm.
Protein may need glycosylation to act
May kill host-bad for yield
Localising proteins (3 methods)
- GFP+ its colour variant proteins fused in for direct visualisation or for antibody recognition (immunoprecipitation/ immunoblot). Different fluorophore colours producing a third colour shows co-localisation of multiple proteins.
- Epitope tags (e.g., flag, Myc or HA) easily recognised by antibody, Western blot helps visualise levels of tagged protein present. NB localisation achieved by separately Western blotting, e.g., nuclear (Lamin A/C ctrl) and cytoplasmic (Tubulin ctrl) fractions, with ctrls confirming separation is clean.
- Reporter genes attached downstream to regulatory seq of gene of interest. Include, e.g., GFP, GUS. Beta-galactosidase w/ X-gal staining, Luciferase (firefly or renilla), CAT. Help analyse (/visualise) level of promoter activity in different tissues. Also analyse effect of other regulatory seqs, e.g., miRNAs
IDing interacting proteins: 3 methods
Immunoprecipitation: Antibody recognises protein/ fused epitope tag (can also use antibody specific to protein of interest but less efficient at precipitation and more chance of non-specificity)-> Antibody-protein complex (immunoprecipitated) isolated with A or G beads- may be magnetic-> run SDS-PAGE, identify co-precipitating proteins with mass spec-> immunoblot to verify protein-protein interaction.
Pull-down assay: only for soluble proteins. Immobilise tagged protein on column-> add mix of potentially interacting proteins through column-> wash unbound protein-> elute-> SDS-PAGE and mass spec ID interacting protein-> verify interaction with Western.
Yeast two-hybrid system: Screen gene library of unknown proteins for interaction. Most commonly use Gal4-> activates gene downstream when DNA-binding domain and transcription activation domain joined, either directly or by pair of interacting proteins-> use latter property: fuse 2 query proteins to each of the domains (see fusion proteins)-> if downstream reporter gene inactive, proteins don’t interact (as Gal4 won’t work)+ vice versa. Somewhat prone to false +ves/-ves.
Site directed mutagenesis for Identification of important residues/domains/regulatory seqs
Site directed mutagenesis: Point mutations/small indels: anneal chemically synthesised oligo with mutation to cloned gene copy, complete strand, transform into E coli, which will correct or go along with mutation- possible to bias toward mutant selection. Also using PCR- instead of mutated oligos use mutated primers-> high fidelity Pol often needed, and always put mutation in primer middle (at least 10 correct bases either side), with Tm>78
Tm= 81.5+ 0.41(%GC)- 675/N- %mismatch, where N=primer length and %s rounded to whole numbers. %GC>40 and primer should end in G/C.
Incorporation and extension of mutagenic primers gives nicked circles-> digest non-mutated, methylated parental strand with DpnI (targets methylated/hemi-methylated DNA, like the E-coli dam methylated stuff) transform new nicked circle into E coli, where nick ligated.
Always sequence the product- PCR could have introduced more mutation!
Analysing function of individual domains
Analyse function of individual domains: Amplify and clone each separate domain (only possible if individual domains encoded by contiguous sequences); ensure domain boundaries are appropriate to avoid misfolding/aggregation (domain prediction programmes/ structural studies/ structure of analogous proteins help); note that individual domain may behave differently in and out of context of whole protein
RNAi: workflow and troubleshooting
RNAi: specifically target cells/organisms. Triggered by dsRNA (processed into guide strand and passenger strand siRNAs by Dicer, a RNase II ribonuclease). siRNAs double stranded, 2nt overhangs at 3’, Pi at 5’, ~21nt. Choice of guide depends of stability of 5’ ends, as passenger is destroyed.
siRNAs assemble-> RISC (RNA induced silencing complex). Guide strand active for specific targeting of mRNA (perfect complement)-> duplex recruits Ago2 which cuts target (endonucleolytic cleavage between nts 10+11 upstream of 5’-most base pair)-> mRNA degradation completed by native pathways.
NB in mammalian cells long dsRNA can induce cell death via interferon response, so siRNAs used to induce RNAi instead by direct transfection using Lipofectamine-3000 (catalytic liposome formulation, complexes w/-ve nucleic acid to overcome electrostatic repulsion of cell membrane); or, plasmids express shRNAs-> transfect into mammalian cell, processed there like miRNAs (see above), expression transient or stable, constitutive or inducible.
Western blot/ RTqPCR verifies success and extent of knockdown (consider long half lives! Still around even when no corresponding mRNA)
If knockdown insufficient: try SMART pool of RNAis, increase siRNA concentration, target a different region with siRNA, try extending the siRNA to increase binding specificity. If all else fails, try a different technique, like mutagenesis or CRISPR knockout (below)
CRISPR/Cas9 workflow
CRISPR/Cas9: permanent+ complete downregulation or mutation. Uses Cas9 nuclease (dsDNA endonuclease- ds breaks repaired by non-homologous end-joining (NHEJ) or homology directed repair (HDR)), target complementary CRISPR RNA (crRNA), auxiliary transactivating crRNA (tracrRNA). Cr and tracr expressed together as guide (g) RNA that mimics natural cr/tracr chimera in bacteria. crRNA designed with software, search engines help find best crRNA and give info on likelihood of off-target effects. crRNA clones into specific site on CRISPR plasmid to produce gRNA that pairs target DNA. Plasmid also encodes Cas9, and possibly additional seqs for cell selection. E.g., Invitrogen pre-linearised plasmid with 5bp overhangs and specific gRNA, Cas9 with nuclear localisation signals, and selectable marker encoded.
Cas9 variants can improve efficiency and specificity, reduce off-target effects, deliver effector molecules and fluorophores. May utilise PAM seq (Cas9 from different organism), dead Cas9 to create targeted roadblock/specifically recruit another effector protein/fluorophore.
Homologous recombination and floxing
Homologous recombination: Knockout endogenous gene by switching with a mutated one: clone gene to be disrupted, inactivate with insertion of selectable marker, insert into host on non-replicating DNA, select for marker (only successfully recombined cells).
Controlled excision/floxing: use cre-lox method (from bacteriophage). Replace target gene with copy flanked with lox seqs (homologous recomb, see above), add Cre (can express only in certain tissues by putting downstream of tissue-specific activator- especially if total knockout lethal), Cre mediates recombination across lox to excise gene.
DNA microinjection for transgenic organisms
DNA microinjection: inject pronucleus of fertilised eggs at single cell stage, DNA randomly integrates, some inserts in site where expression enabled, often used to insert new info/cause overexpression. Cell transplanted into foster mother’s oviduct, 10-20% pups transgenic (e.g., growth hormone in mice).
Cultured embryonic stem cells/ making transgenic organisms (workflow)
Cultured embryonic stem cells: manipulate in lab, then re-inject into embryo, where some cells incorporated to form mosaic organism. Often used for knockout:
Target gene replaced by positive selection marker (e.g., antibiotic resistance)- could also make subtle changes to target gene. Introduce DNA constructs, homologous recombination with target gene: floxed cassette inserted, disrupted allele introduced, select for double crossover integrating floxed gene, Cre added for specific tissue disruption. Cell selection using integrated markers (-ve selection markers may be used to ensure proper recombination (die if not recombined properly). Individual transformant cultured, analysed to ensure proper DNA integration, injected into developing embryos.
Resulting mosaic organisms can be mated to non-transgenics to get fully transgenic, heterozygous progeny (knockout mice)- can cross further for homozygotes (if not lethal)
