Cloning Flashcards
What is a vector?
Self-replicating DNA that is independent of the chromosomal DNA. E.g. plasmids. Also called episomal DNA
What is cloning?
The insertion of a fragment of DNA into another DNA molecule that can self-replicate in order to produce more of the fragment DNA.
What must all cloning vectors have?
Origin of Replication
Selectable Markers
Multiple cloning sites
What is the ORI?
Origin of replication is the region of DNA recognised by the cellular DNA replication enzymes. Without it, the recombinant molecule cannot be replicated
What are Expression Vectors?
They are vectors that allow a cloned segment of DNA to be translated into a protein inside a bacterial or eukaryotic cell
What are the 4 things that expression vectors contain?
Ori (appropriate for expression host)
Selection marker (Ab marker)
Promoter (prokaryotic or eukaryotic)
N- or C-terminal tags or fusion protein for purification
What is the function of expression vectors?
They produce large amounts of a specific protein, which allows the study of structure and function of proteins. Can be useful when proteins are difficult to isolate or are rare cellular components.
What is the function of the promoter in expression vectors?
To allow the expression of the cloned gene
What are the two types of expression vectors, and their associated promoters?
Mammalian and Bacterial expression vectors.
Strong viral promoter (from CMV) for mammalian and strong phage promoter (T7 promoter) for bacterial.
What are Reporter Gene Vectors?
These are vectors used to study promoters and the transcriptional activity from regulatory sequences. The genes in this case express a protein that can be quantified eg. luciferase/B-galactosidase. Any changes in their expression can be attributed to the function of regulatory sequences.
What are shuttle vectors? What are their advantages?
They are vectors that can replicate in more than 1 organism, eg in mammalian and bacterial cells. They can be used to manipulate DNA in different cell types, eg. clone a human gene in bacteria, and test its function in yeasts/animal cell
What are the 3 types of cut ends produced by restriction enzymes?
5’ overhang
3’ overhang
blunt ends
What are isoschizomers?
Pairs of different REs that are specific to the same recognition sequence. E.g. SphI (CGTAC/G) and BbuI(CGTAC/G)
How do you fill in the 5’ and 3’ overhangs to form a blunt end?
Use Klenow fragments for 5’ overhang
Use T4 DNA polymerase for 3’ (exonuclease activity “chews back” in presence of dNTPs)
both need dNTPs
What can be done to minimize self-ligation if vector is cut using 1 RE, or esp with blunt ends?
Use alkaline phosphatase or calf intestine phosphatase. This removes the phosphates from vectors after they’re cut, and since ligases need phosphate and OH, this can’t happen anymore, therefore preventing self-ligating. Since insert still has 2 P, it will ligate.
What are the two ways to determine the orientation of the insert or to ensure directional cloning?
1- determine orientation by digestion of recombinant vector.
2- clone using heterologous ends, ie use 2 different REs for both insert and vector
What are the 3 advantages of directional cloning?
1- improved efficiency
2- ensure directional cloning
3- low background of non-recombinants
How do you generate a new RE site from a blunt end?
Add a linker, ie a ds DNA that contain a RE site, eg. an EcoRI site
Digest using RE to produce sticky ends
NB to ensure that RE sites are not right at ends of linker
What is the significance of the 3:1 Insert to Vector ratio?
To prevent vector ligating/closing on itself.
What are the 3 methods in which bacteria transfer/exchange their genetic material?
Conjugation- joined via pilus and locked membranes
Transformation - uptake from the environment
Transduction - via phages
What are the applications of cloning?
1- Construction of genetic libraries assist in isolation/characterisation of specific genes and gene products
2- Production of scarce proteins using expression vectors
3- Mass production of vaccines and hormones
4- Diagnostics- produces Ab and oligonucleotide probes
5- Therapeutics- human insulin, human growth hormone etc
6- Gene transfer (eg drought-resistant plants, gene therapy)
What are the basic steps in cloning?
DNA fragment + Vector + DNA ligase = Recombinant DNA molecule + bacterial host = cloned bacteria = Ab selection
List the 3 types of inserts.
1) . Genomic DNA inserts
2) . cDNA inserts
3) . PCR amplified inserts
Outline the 4 strategies for cloning PCR products ie PCR amplified inserts.
1- Blunt-end cloning: proof-reading DNA polymerase eg Pfu generates blunt ends. Clone into blunt-end vector
2- Restriction site end cloning: Introduce RE target sites into the primers for PCR, then digest PCR product and vector with RE
3- T/A cloning: non-proofreading poly eg Taq has terminal transferase activity, adds 1 or 2 A to end of product. Must use T-tailed vector
4- Site-directed mutagenesis: Introduce nucleotide (codon) changes into insert
What is the function of the selectable marker in the vector?
To make the plasmid ‘useful’ to the cell, thus allows it to be maintained in the cell
Only cells with these markers eg Ab resistance, can survice
What are the types of vectors relating to the size of DNA to be cloned?
Plasmid - up to 10kb
Phage vector - 12-20kb
Cosmids - 40-45kb
Artificial chromosomes (eg BAC, YAC, PAC) - over 100kb
List the 2 disadvantages of plasmid vectors
1) . Limits the insert size
2) . Standard transformation methods in E. coli inefficient, therefore need Ab resistance selection
Define “competency”
The ability of a cell to take up extracellular “naked” DNA from its environment
What are eg of naturally competent bacteria, and how can artificially induce competence in bacteria?
naturally-competent: Bacillus, haemophilus, streptococcus
To induce: treat with CaCl2, Rubidium chloride, DMSO
What are the 5 advantages of using E. coli as the host bacteria?
1- genetic simplicity 2- Safe 3- Genome sequence known (expression of foreign proteins) 4- Ability to host foreign DNA 5- rapid growth rate
How do you calculate transformation efficiency?
TE = no. of colonies per ml / [DNA] (ug/ml) transformed
= no. of colonies formed per ug of DNA
Good when 10^8 transformed, poor when <10^4
List the types of Controls in cloning
Ligase control Transformation control Cell viability Contamination control Linear DNA control
Describe the principles behind blue-white screening and alpha complementation
This method is used to see whether the insert is taken up into the vector. The cells with vectors with recombinant DNA will form white colonies while those with vectors without insert will form blue colonies.
The principle lies with Beta-galactosidase, an enzyme that cleaves X-gal, causing blue pigmentation. B-gal is coded by the lacZ gene. There is a mutant B-gal derived from M15 strain of Ecoli that has its N-terminal deleted, therefore is inactive. This part is the lacZ-a, and the enzyme becomes active after it rejoins the “w” part (rest of enzyme). To exploit this, the bacteria host with the mutant B-gal (ie the M15 Ecoli) is used, and the plasmid has lacZ-a in its MCS, where the insert replaces upon ligation. If the vector (plasmid) doesn’t have the insert, the cell colony after transformation would be blue, as the B-gal is complete (fusion of the chromosomal-translated and the plasmid-translated parts), and thus would cleave the X-gal. If the plasmid has the insert, the colonies would be white.
