Recombinant DNA technology I Flashcards
What is cloning? What is molecular cloning?
Cloning = make many identical copies of
* an organism
* a molecule
PCR
Restriction enzymes
restriction endonucleases
Bacteria transformation
Plasmids
These 4 came together to allow for molecular cloning
Overview of molecular cloning
- Obtain the gene of interest GoI (or region of interest RoI) (extract DNA, PCR)
2.“Cut” GoI and vector with appropriate restriction enzymes
(REs) so that they can be joined (REs) - Separate and isolate fragments (agarose gel electrophoresis)
- Prepare the plasmid (cut or PCR amplify it)
- Ligate (join) GoI (or RoI) and vector together (ligase)
- Insert the new, recombinant, molecule, in bacteria
(transform) (recipient bacteria, plates) - Grow clonal bacteria in a selective environment, select
for growth of the transformed bacteria - Grow the bacteria (single colony)
- Screen (check) bacteria for the (correct) insert (PCR, REs,
sequencing, visual) - Correct clones can be cultured on a large scale, yielding
pure DNA of the correct sequence
Why go through all these steps instead of just PCR
vector ar more susceptible to be broken
if you freeze just DNA it is likely to be degraded
if it is in the bacteria, they are more stable and if you freeze them they can last a lot longer without DNA being degraded and you might not need primers, grow bacteria, extract plasmids and cut so you don’t need plasmids
insert might be quite long which is hard to amplify
Enzymes for PCR
how to choose a polymerase?
Characteristics of DNA polymerases
* processivity (number of nucleotides a DNA pol can incorporate before it dissociates from template) (how long is the run of nucleotides that can be synthesised)
*fidelity (how error free it is) (proofreading, 3′→ 5′ exonuclease activity)
* specificity (background amplification, HotStart PCR)
*thermostability (half life at a specific temperature) (how quickly does it degrade)
Types of DNA polymerase enzymes for PCR
(dont memorise)
- Taq polymerase (from Thermus aquaticus)
- Optimal temperature for DNA synthesis 72-75°C
- 5’→3’ DNA polymerase activity but no 3’→5’ exonuclease activity => no proofreading
- short half-life at 95°C
protein may be different if there are errors
Proofreading polymerases (with 3’→5’ exonuclease activity), e.g.
- Pfu DNA Polymerase (from archaeon Pyrococcus furiosus) slower than Taq DNA Polymerase but proofreading
-Platinum® Taq DNA Polymerase High Fidelity
* higher fidelity than Taq (Platinum® Taq DNA Pol + proofreading 3’→5’ exonuclease activity enzyme)
less mistakes likely
- Phusion® High-Fidelity DNA Polymerase
- a novel enzyme fused with a processivity-enhancing domain
- error rate much lower than Taq DNA Pol
- Q5® High-Fidelity DNA Polymerase - high-fidelity and thermostable
- error rate much lower than Taq DNA Polymerase and Pfu DNA Polymerase
Some common types of PCRs
- HotStart PCR, short time at the beginning to denature an inhibitor (antibody) when you heat this antibody it is not resistant so it’s denatured so polymerase becomes denatured.
- Touchdown PCR = decreasing annealing temperature in order to have high specificity to then go down in specificity. First few cycles not much quantity but highly specific so good quality then after at lower temperature just to increase quantity
- Nested PCR= first PCR with non specific primers then second PCR with higher specificities as you have more copies more likely to have more specific at higher quantities.
What can cause problems with PCR products
loads of c-g
epigenetics
long fragments /targets
in solution molecules may interact with strange artefacts
Gel electrophoresis
- DNA is negatively charged
- Smaller fragments run faster in agarose/acrylamide gel
- Visualise DNA with staining (EtBr, SYBR stains, etc.)
physically cut agarose with that DNA
Restriction enzymes (endonucleases)
- originally a form of bacterial immunity
against viruses - bind to DNA at specific sequences (often
palindromic) and cut them - names from the bacterial species they were
isolated from, and a sequential number - the cut can have blunt ends or “sticky” ends
Plasmids
- small circular extra-chromosomal DNA
- multiple copies per cell
- usually contain few genes:
- genes to replicate independently from the bacterial genome
(ori, replication control, etc.) - genes to enable cell-to-cell transfer of plasmid DNA
- genes to modify host phenotype (e.g. antibiotic resistance)
- modern plasmid are highly engineered to perform these
functions very efficiently (plus integration of DNA
The plasmid pUC19 Important elements:
- origin of replication
- selectable marker
- multiple cloning site (MCS)
- blue/white colony screening
Horizontal gene transfer
Transformation (transfection eukaryotic cells) - transduction - conjugation.
Bacteria transformation
Transformation (transfection) of bacterial cells to make
“competent” cells, slightly modified bacteria to make them prone to pick up DNA
Ligation
DNA ligase repairs DSBs (double strand breaks) in DNA so can be used to manually “link/paste” together two pieces of DNA
Blunt ends or sticky ends
Depending of the restriction enzyme, or on the DNA polymerase you used
* The restriction enzyme HpaI
* Platinum® Taq DNA Polymerase High Fidelity
* Phusion® High-Fidelity DNA Polymerase
* Q5® High-Fidelity DNA Polymerase
generate blunt ends
proofreading polymerases generally create DNA with blunt ends.
* The restriction enzyme EcoRI
* Taq DNA polymerase
generate sticky ends
