What is the ‘full’ definition of cloning?
The isolation of a particular nucleotide DNA sequence from its genomic context and the production of multiple identical copies of that sequence
What are three uses of cloning?
- Isolation of genes or gene fragments for in vitro study
- To obtain large quantities of a protein encoded by a particular gene for in vitro study or medical purposes (eg. insulin)
- Perpare modified versions of genes for reintroduction into the original host for functional studies
What requires vectors, hosts and DNA manipulation?
Cloning
What are the broad steps of cloning? (4)
- Foreign DNA fragment is prepared by restriction or PCR for insertion into a vector
- The fragment is inserted and ligated into the vector to make a recombinant DNA molecule
- These recombinant DNA molecule is inserted into host cells and the cell culture is heat shocked or electroporated
- Some sort of selection for cells containing recombinant DNA molecules occurs
What are used for cloning relatively small inserts? (less than 10 kbp)
Plasmid Vectors
How big are plasmid vectors?
Around 1-200 kb in size
What are three essential features of plasmid vectors?
- Have an origin of replication (ori) site
- Have selectable markers (to distinguish cells with the plasmid from those without)
- Unique restriction enzyme cleavage sites (most located in MCS)
What type of vectors are used to clone inserts around 20 kbp?
Bacteriophage cloning vectors (eg. lambda genome)
What type of vectors are used for 100-300+ kbp fragment cloning?
BACs (Bacterial artificial chromosomes)
BACs are propagated at high or low copy numbers in E. coli?
low copy numbers
What type of vector is used to clone ‘linear DNA fragments’ up to 2000 kbp?
YACs (yeast artificial chromosomes)
What are three components of YACs (yeast artificial chromosomes)?
- Yeast replication origin (ori)
- Centromere (CEN)
- Telomere (TEL)
How do YACs come? How must they be treated to allow for fragment insertion?
YACs come as circular chromosomes. They contain two telomere sequences bounded internally by BamHI cleavage sites. Cleavage by BamHI makes a linear molecule with telomeres at two ends.
Then EcoRI digestion must occur to cleave X and Y markers from each other and genomic DNA is inserted and ligated between these. The recombinant YAC is then ready to transform yeast cells.
How are YACs inserted into yeast cells?
- The yeast cell wall must be enzymatically degraded
- The resulting yeast spheroplast is then susceptible to transformation from YAC
What type of restriction enzymes can be used to generate cloning compatible ends?
Type II restriction endonucleases
How can PCR reactions add restriction sites to molecules?
By adding restriction sites to 5’ ends of the PCR primers
How are restriction enzyme linkers (aka adapters) added to blunt end restriction endonuclease products?
With T4 DNA ligase
What is TA cloning?
Takes advantage of the fact that DNA polymerases (eg. Taq polymerase) can add a single 3’ A residue to blunt ended double stranded DNA
- PCR products or other A tailed products can therefore be ligated (inserted) with DNA ligase into a T-tailed vector
What is a T-tailed vector?
A linearized vector with 3’-T overhangs
Can have A tailed products ligated (inserted) in
What is TOPO-TA cloning?
TA cloning (inserting A-tailed products into T-tailed vectors), but with a topoisomerase covalently attached to the vector. The 5’ OH group of a A-tailed PCR product can attach this bond and release the topoisomerase I enzyme.
Take home message is that DNA ligase is not needed. The vector come pre-prepared, just add the A-tailed product (from Taq polymerized PCR etc.)
What is one method of directional cloning?
- Use a restriction enzyme that makes sticky ends for one end of the insertion and a another restriction enzyme to make a blunt end at the other end to digest chromosomal DNA for insertion
- Can insert into a plasmid cloning vector cleaved with the same restriction enzymes
What type of plasmid is used in blue-white screening? What are it’s three main features?
A pUC8 plasmid
- Ampicilin resistance gene
- lacZ gene codes for beta-galactosidase enzyme
- Has a MCS within lacZ open reading frame
How does the blue-white screening test work?
Because the pUC8 plasmid used has a MCS within its lacZ gene, those with an insert will not produce a viable beta-galactosidase. When plated with X-gal sugar, the non-recombinant host will split x-gal into components, one of which is blue. The recombinant host cannot split x-gal and therefor is white.
Does a blue product from blue-white screening indicate recombinance or no recombinance? Why?
No recombinance. Because blue product is the result of x-gal digestion by beta-galactosidase, which is not produced when a recombinant insert is in the lacZ gene of the pUC8 plasmid.
