What is Gene cloning?
The process in which the gene of interest in linked to a vector that enables its amplification and propagation as a pure population of molecules in cells
What is molecular cloning of DNA is important to?
- Purify and amplify genes of interest
- Obtain their DNA sequences
- Determine the gene structure and regulation
- Perform site-directed mutagenesis to investigate function
- Express and purify protein for biochemical/structural analysis
- Enable genome analysis by creating overlapping clones of genomic DNA
- Reintroduce genes into another organism i.e. transgenesis
What are terminal transferases?
Add homopolymer tails to the ends of DNA
What are polynucleotide kinases?
Add a phosphate to the 3’ end of DNA
What are alkaline phosphatases?
Remove terminal phosphates from DNA end
Why do bacteria use methylases?
Bacteria can restrict bacteriophage infection by using the appropriate endonuclease to recognise the bacteriophage’s DNA and degrade it
However, to protect its own DNA, bacteria will express cognate methylases to modify its DNA.
Presence of methylation confers cleavage so that the bacteria does not degrade its own DNA
Explain the binding of BamHI to its recognition sequence
- BamHI restriction endonuclease binds as a homodimer and forms a 2-fold symmetric enzyme DNA complex
- Enzyme slides along the DNA helix
- When the recognition sequence is encountered, the conformation is altered and results in a tighter atomic interaction
- Tighter interaction causes the cleavage of both strands
What are the three types of termini are produced by Type II restriction enzymes?
3’ recessed ends
Blunt ends
5’ Recessed ends
What groups do the termini have after use of the restriction enzymes
5’ termini of each strand in the cleavage products have Phosphoryl group
3’ termini of each strand in the cleavage product have Hydroxyl group
What is the function of DNA ligase?
Catalyse the formation of 5’ - 3’ phosphodiester bonds in double-strand DNA molecules between juxtaposed 5’ phosphate and 3’ hydroxyl
Ligase requires a co-factor that forms a covalent intermediate with the enzyme
How does the concentration of DNA in ligations affect the outcome of the products?
Higher [DNA] results in intermolecular ligation to produce linear concatenated DNA
Lower [DNA] results in intramolecular ligation to produce circular DNA (covalently closed)
At what concentration of DNA would the desired insert be added into a vector DNA?
Low [DNA] to give a covalently closed circular products
What is meant by the processivity of a DNA polymerase?
The ability of DNA polymerase to carry out continuous DNA synthesis on a template DNA without frequent dissociation.
It can be measured by the average number of nucleotides incorporated by a DNA polymerase on a single associated/dissociation event
Name the types of DNA polymerase (4)
E.coli DNA polymerase: has both 3’ to 5’ and 5’ to 3’ exonuclease activity. Has 5’ to 3’ synthesis activity
Klenow sub-fragment of DNA polymerase: lacks 5’ to 3’ exonuclease thus used for initiating DNA synthesis from oligonucleotide primers for radioactive labelling or chain terminator DNA sequence
T4 DNA polymerase: has 5’ to 3’ synthesis activity. High levels of 3’ to 5’ exonuclease activity thus useful for trimming restriction fragment ends
T7 DNA polymerase: has very high processivity and is used for chain terminator DNA sequencing
How is Klenow DNA pol used to make radioactive DNA probes (3)?
- The DNA molecules is denatured and hexanucleotide primers are annealed
- Klenow DNA pol and radioactive dNTPs are used to synthesise a labelled DNA strand until it reaches the next primer
- DNA is denatured and the probes are separated into their respective fragments
What is the function of T4 polynucleotide kinase?
Catalyse the transfer and exchange of phosphate from the gamma position of ATP to the 5’ hydroxyl terminus of double and single-stranded DNA and RNA
How can polynucleotide kinases be used?
To efficiently label the 5’ end of restriction fragments or synthetic single-strand oligonucleotides to use as probes using γ-32P-dNTP as the radioactive nucleotide
To phosphorylate PCR products made with non-phosphorylated primers ready for ligation
What are the common properties of a cloning vector?
- Ability to promote autonomous replication and amplify from a single copy
- A genetic marker(s) to select for or identify cells containing the vector
- Unique restriction sites to facilitate cloning of insert DNA
- Minimal non-essential DNA to maximise size range of cloning
How can self-ligation of the vector be prevented?
Alkaline phosphatase are used to remove phosphate groups from the vector ends. Therefore, the ligase is unable to form phosphodiester bond between the vector ends.
Vector and Insert can still ligate together because the insert ends have phosphate groups.
How can DNA molecules derived from different restriction enzymes be linked (4)?
- Fill in or resect DNA ends with T4 DNA polymerase plus dNTPS and perform blunt end ligations
- Add by blunt end ligation duplex linkers containing desired site and then cut with restriction enzymes to create cohesive end
- Add by blunt end ligation adaptors with appropriate performed cohesive end (no restriction enzyme cutting then necessary)
- Add desired restriction enzyme sites by PCR using oligonucleotide primers with restriction sites in 5’ end of oligonucleotide sequence and then cut with restriction enzymes to create cohesive ends
Explain the lytic pathway of λ Bacteriophage
- Lambda DNA circularises for replication
- Catenane “rolled off” the λ DNA molecule
- Ter enzymes generates staggered nicks at the Cos site
- Genome packaged into phage heads
Sometimes this goes wrong and E.coli DNA is packaged instead
Explain the in vitro process of λ DNA packaging into phage particles
Require two lambda strains -> 1 with mutation in pE (no head formation) and 1 with mutation in pD (no packaging). One set of bacteria infected with one lambda defect and one set with the other
Next, lyse the bacterial cells and take the lysates out. Mix lysates with recombinant lambda DNA (gene of interest).
They complete each other in vitro –> complete lambda structure
How are λ converted into cloning vectors?
Non-essential region for lytic infection is deleted. This will allow insertions into λ of up to 10 Kbp
How are λ replacement vector generated?
Non-essential region can be deleted entirely and substituted with a ‘stuffer’ region containing restriction enzymes sites to create a replacement vector
