DNA analysis and manipulation Flashcards
What are some reasons why we need to manipulate DNA ?
- To help produce recombinant protein (or RNA!) for biochemical / structural analysis
- To sequence DNA (e.g. mutant analysis)
- To complement mutants – linking genotype to phenotype
- To be able to construct specific mutations to investigate gene/enzyme function
- To make fusions to reporter genes – e.g. GFP
- To generate new biochemical pathways and regulatory circuits in bacteria, plants and animals
What are functions of plasmid vectors ?
- General DNA cloning and manipulation (e.g. pBluescript II)
- DNA sequencing
- Protein over-expression e.g. pET21
- Gene expression – complementation (organism-specific)
- Gene expression – reporter genes (organism-specific)
Explain pBluescript II ?
- A modern cloning vector
1. Ampicillin resistance gene
2. pUC origin of replication
3. Lac operon promoter
4. Multiple cloning site
5. lacZα fragment gene
6. Phage f1 origin of replication (rarely used)
Name some examples of restriction enzymes ?
- AluI
- EcoRI
- KpnI
Gene amplified by PCR using ?
Chromosomal DNA or cDNA
What is the PCR product cloned into ?
- Standard cloning vector and sequenced (to check for mutations)
- Gene moved to different vectors – e.g. for over-expression
Extent of DNA is defined by ?
PCR primers
What do PCR primers not need to do ?
PCR primers do not need to anneal to the
template DNA at the 5’ end
Engineering genes for easy protein purification?
- Mix cut vector and insert
- Add DNA ligase
- Transform E. coli
As long as there is sufficient complementarity at the 3’ end. What will an oligo do ?
An oligo can be
extended > 40 nt upstream to incorporate restriction site, ribosome binding site
etc.
What do restriction enzymes often not do?
Restriction enzymes often do not cut close to DNA ends, therefore DNA is often blunt-end cloned first, then subcloned
What is isolated plasmid usually a mixture of ?
Covalently closed circular (CCC) and open circular (OC)
Restriction enzymes cut?
One strand of DNA first (i.e. make OC)
What do Partial digests give ?
Greater than expected bands
Explain Inverse PCR mutagenesis ?
Once a gene is cloned you can make a mutant allele (e.g. deletion or single amino acid) then reintroduce this into the wild-type strain by homologous recombination to generate a mutant. The mutant protein can also be overexpressed and studied
Inverse PCR mutagenesis uses circular template and divergent primers, used for ?
- Amino acid substitutions
- Large deletions
- Small insertions
Who invented Gibson assembly ?
Invented by Daniel Gibson at Craig Venter Institute
What does Gibson assembly allow to happen ?
Allows simultaneous assembly of several DNA fragments with overlapping (~20 bp) ends
What does the Gibson assembly not require ?
- Restriction sites
- No scars are left (seamless)
- Specialised plasmid vectors (unlike Golden Gate cloning)
What kind of reaction and process is Gibson assembly ?
- One tube reaction
- Isothermal (PCR not involved in the actual assembly)
What is needed for Gibson Assembly ?
Add (at 50°C):
- T5 exonuclease
- Phusion polymerase
- Taq ligase
What is the aim of Gibson assembly ?
You aim to assemble 3 DNA fragments each containing a gene or part of a gene into a plasmid in one step
In Gibson Assembly your fragments will usually be either ?
- In Gibson Assembly your fragments will usually be either PCR products or
synthetic DNA fragments (synthesised using oligonucleotides by a company) - This means the ends of the DNA fragments can be customised
PCR products can be produced with ?
Long 5’ tails (e.g. 30 nt tail)
