Saul Purton Molecular Biology Methods Flashcards
What was the traditional method to amplifying DNA prior to PCR?
What are the 3 steps to PCR?
3 Things why PCR is made possible
What is the optimal, maximum rate of PCR?
Why is maximum rate not 100% achievable in the exponential phase?
What happens during the plateau phase?
What is the PCR threshold?
Cloning DNA into a vector & inserting into host for over-expression
- DNA denaturation ~95C
- Primers annealing to DNA ~50C
- Elongation of primers by DNA polymerase ~70C
Thermostable DNA polymerase, low cost synthesis of primers, programmable thermocyclers
Product doubles every cycle - unlimited substrates & fully active enzymes
Requires primers anneal perfectly 100% time, and strand synthesis is never 100%
Substrate runes out, polymerase denatures, endless cycles doesn’t mean more products
Where concentration of PCR product enough for detection
For the following primer design considerations, why should it be?
- Optimum length 18-24 bases
- GC content 40-60% with GC at start & end
- Avoid complimentary primer sequences
- Tm of primers similar (<5C diff) and between 60-75C
- Primer tail on 5’ end
- Long enough for specificity but short enough to bind efficiently & requires 3’OH
- Primers bind firmly to limit falling off
- Prevent primer dimer formation
- Tm temp which achieve 50/50 annealing:deannealed - want similar temperature
- To incorporate specific sequences (e.g EcoRI) at end into PCR product as primers become part of the product
What is the problem with primer dimers?
Why are shorter primers more favourable?
What happens if the annealing temperature is too low?
Higher?
Why is a higher temperature more favourable?
End up into PCR product & extended by DNA polymerase so become template for next round of PCR - end up with undesired product
Polymerase can fall off - so if primers are closer together = higher success rate
Formation of other unwanted products like dimers
Smaller bands of target products but fewer unwanted products
Retains specificity of primers to bind effectively, and end up with more efficient PCR of target product (and fewer unwanted ones)
Why is a smaller volume of PCR tube with thinner wall more favourable?
Why is a heated lid on a thermocycler more favourable?
What is the problem with PCR & its use in detection/analysis?
What do you need to consider in PCR for cloning?
How can PCR be used in site-directed mutagenesis?
More efficient heat transfer
Stops evaporation & ensures all tubes are the same temperature
High sensitivity - need to use controls to see a valid result
5’ tails to introduce restriction sites
Recombination of PCR products by primers binding to a common sequence & overlapping- then amplification of the recombination product
How can you use RT-PCR to produce DNA products from mRNA?
What is the problem with the DNA product?
How can you tackle the contamination of genomic DNA in the cDNA sample?
How can you quantitate PCR?
What is the level of fluorescence proportional to?
What is the Cq value?
How can you determine product concentration from this?
Use reverse transcriptase instead of DNA polymerase to make cDNA copy, then amplify cDNA to make it double stranded, then amplify that in PCR
mRNA has no introns - so neither will the DNA (smaller)
Designing primers across exon boundaries/using DNAse to degrade genomic DNA before reverse transcription in mRNA sample
qPCR & using fluorescently labelled primers that only fluoresce once incorporated into the PCR product
Amount of PCR product/amplicon
Cycle of PCR where fluorescence becomes detectable above background levels
Compare Cq value with standard dilution curve with Cq values at known concentrations
What 3 features are important for plasmids in recombinant technology?
What is desirable?
What is required for an expression vector?
How can you improve expression?
What is crucial for plasmids expressing toxic proteins?
Ori, selectable marker, insertion sites (MCS) to not disrupt other genes
Small size, high copy number, selection/screen (lac system)
Promotor, ribosome binding site, terminator
Protein tag for antibody detection/protein purification, binding proteins for purification, protease site to remove tags
Tight control/inducible promoter system to maximise protein production before cell death
How is the T7 promotor system induced in pET vectors?
How is the host engineered?
How does the inducer show tight regulation of gene expression?
What happens when inducer is not present?
IPTG
To contain T7 system & produce T7 RNA polymerase
T7 RNA polymerase only transcribes in presence of IPTG
Repressor remains bound to operator & no transcription from promoter
What are the 2 problems with plasmid vectors to make libraries?
Why is lambda’s genome successful as a vector?
What else makes it successful? 3
What is lambda’s problem?
- Cloning capacity (unstable with >10kb insert)
- E. coli transformation efficiency (need to be competent)
Has 20kb central region of lysogeny genes which are not required for lysis - so can be replaced
- Insert larger amounts DNA (restricted to head size)
- Packaging of DNA performed in vitro using two packaging extracts (defects in packaging lacking essential protein)
- Delivery is efficient to E. coli as it’s infection
Has 5 EcoRI sites - only want 1 in non-essential region
What are the 5 steps to inactivating these sites?
What is the selective advantage?
What are the 3 steps to DNA cloning in phages?
How can you produce libraries from this?
- Phage DNA injected into E. coli carrying RI plasmid allow modification of phage DNA
- Only Phage DNA that has been methylated prior to restriction survives & give rise to progeny (lytic cycle) ~ 1%
- Progeny with methylated EcoRI sites infect & inject DNA into E. coli without RI plasmid ~100% survival as no restriction enzymes present
- Progeny with EcoRI sites are unmethylated - reset without presence of restriction modification system
- Progeny re-insert into E. coli carrying RI plasmid & cycle repeats - selecting for mutants that have loss EcoRI sites
More EcoRI sites = higher chance of death due to methylation, so selective advantage to have mutations deleting the sites so phages are not affected by methylation & survive
- restrict phage DNA with BamHI which removes non-essential region
- restrict & ligate desired DNA fragments & seal with DNA ligase
- recombinant phage DNA can be packaged in vitro using phage-assembly system into virions containing recombinant DNA
Transform virions into E. coli & plate onto agar - pick clones from plaques (as they contain recombinant phage) & then re-infect with E. coli
What is a cosmid vector?
What are the 2 requirements?
What happens on DNA cloning?
So what technique does cosmid DNA transfer use?
Hybrid vector between lambda phage & plasmid
- 2 cos sites (one at start & end of DNA with BamHI restriction site between)
- 48kb separation between sites
DNA packaged into lambda using in vitro packaging assembly & the cos sites - allows for linearisation (rolling circle DNA) into progeny to then infect bacteria
Transduction
What is a fosmid vector?
So what technique does fosmid DNA transfer use?
What does fosmid allow which cosmids don’t?
Low copy number cosmid
Uses F sex factor pilus for conjugation
Use for larger DNA to be cloned <40kb while being stably maintained
What is a BAC vector?
What does it allow on cloning?
What is a YAC vector?
What does it allow on cloning?
What will the vector contain?
Plasmid vector containing F episome with elements for controlled replication & partioning (bacterial artificial chromosome)
Insertion up to 300kb DNA
Same but for chromosome maintenance & replication in yeast nuclei
200-500kb DNA
Selectable markers, ori for yeast & yeast centromeres & telomeres
What was Chargaff’s rule in 1940s?
Watson & Crick’s rule in 1953?
Nirenberg’s genetic code rule in 1966?
What was Maxam & Gilbert’s chemical modification/DNA cleavage method?
1:1 ratio of pyrimidines:purines
Double stranded with AT:GC
Triplet bases of DNA - linear relationship with protein sequence
Treat DNA with chemicals to modify the DNA & cleave
Key points of the original Sanger Sequencing method.
- What polymerase was used?
- What was the 5’ end of the new strand defined by?
- How were the dNTPs labelled?
- How were all possible chain terminations generated?
- How was the DNA sequence determined?
Give 4 problems of Sanger Sequencing
- DNA polymerase/klenow
- oligonucleotide primer
- 32P dNTP or 35S dNTP
- Spiking reaction with ddNTPs in 4 reactions
- Run 4 lanes on polyacrylamide gel & run gel electrophoresis then read from bottom to top
Radioactivity is hazardous & small shelf life
Klenow has poor processivity & stalled at secondary structures
Labelled dNTPs means stalled chains were detected so gave false positive of chain termination
Needed 4 different reactions for each ddNTP
How was radiolabelling improved?
How was reaction improved?
How did Polymerase improve?
How did gel electrophoresis improve?
Use fluorescently labelled ddNTPs (not dNTP) - only true chain termination detected
Done in 1 reaction
T7 DNA polymerase - lacks 3’ to 5’ proof reading, high processivity, faster synthesis, higher efficiency & doesn’t stall at 2ndary structures
Fast capillary system - no gel pouring & automated reading of dyes with laser for computational analysis
