Cloning

Question 0

What is a vector?

Answer 0

Self-replicating DNA that is independent of the chromosomal DNA. E.g. plasmids. Also called episomal DNA

Question 1

What is cloning?

Answer 1

The insertion of a fragment of DNA into another DNA molecule that can self-replicate in order to produce more of the fragment DNA.

Question 2

What must all cloning vectors have?

Answer 2

Origin of Replication
Selectable Markers
Multiple cloning sites

Question 3

What is the ORI?

Answer 3

Origin of replication is the region of DNA recognised by the cellular DNA replication enzymes. Without it, the recombinant molecule cannot be replicated

Question 4

What are Expression Vectors?

Answer 4

They are vectors that allow a cloned segment of DNA to be translated into a protein inside a bacterial or eukaryotic cell

Question 5

What are the 4 things that expression vectors contain?

Answer 5

Ori (appropriate for expression host)
Selection marker (Ab marker)
Promoter (prokaryotic or eukaryotic)
N- or C-terminal tags or fusion protein for purification

Question 6

What is the function of expression vectors?

Answer 6

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.

Question 7

What is the function of the promoter in expression vectors?

Answer 7

To allow the expression of the cloned gene

Question 8

What are the two types of expression vectors, and their associated promoters?

Answer 8

Mammalian and Bacterial expression vectors.

Strong viral promoter (from CMV) for mammalian and strong phage promoter (T7 promoter) for bacterial.

Question 9

What are Reporter Gene Vectors?

Answer 9

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.

Question 10

What are shuttle vectors? What are their advantages?

Answer 10

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

Question 11

What are the 3 types of cut ends produced by restriction enzymes?

Answer 11

5’ overhang
3’ overhang
blunt ends

Question 12

What are isoschizomers?

Answer 12

Pairs of different REs that are specific to the same recognition sequence. E.g. SphI (CGTAC/G) and BbuI(CGTAC/G)

Question 13

How do you fill in the 5’ and 3’ overhangs to form a blunt end?

Answer 13

Use Klenow fragments for 5’ overhang
Use T4 DNA polymerase for 3’ (exonuclease activity “chews back” in presence of dNTPs)
both need dNTPs

Question 14

What can be done to minimize self-ligation if vector is cut using 1 RE, or esp with blunt ends?

Answer 14

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.

Question 15

What are the two ways to determine the orientation of the insert or to ensure directional cloning?

Answer 15

1- determine orientation by digestion of recombinant vector.

2- clone using heterologous ends, ie use 2 different REs for both insert and vector

Question 16

What are the 3 advantages of directional cloning?

Answer 16

1- improved efficiency
2- ensure directional cloning
3- low background of non-recombinants

Question 17

How do you generate a new RE site from a blunt end?

Answer 17

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

Question 18

What is the significance of the 3:1 Insert to Vector ratio?

Answer 18

To prevent vector ligating/closing on itself.

Question 19

What are the 3 methods in which bacteria transfer/exchange their genetic material?

Answer 19

Conjugation- joined via pilus and locked membranes
Transformation - uptake from the environment
Transduction - via phages

Question 20

What are the applications of cloning?

Answer 20

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)

Question 21

What are the basic steps in cloning?

Answer 21

DNA fragment + Vector + DNA ligase = Recombinant DNA molecule + bacterial host = cloned bacteria = Ab selection

Question 22

List the 3 types of inserts.

Answer 22

1) . Genomic DNA inserts
2) . cDNA inserts
3) . PCR amplified inserts

Question 23

Outline the 4 strategies for cloning PCR products ie PCR amplified inserts.

Answer 23

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

Question 24

What is the function of the selectable marker in the vector?

Answer 24

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

Question 25

What are the types of vectors relating to the size of DNA to be cloned?

Answer 25

Plasmid - up to 10kb
Phage vector - 12-20kb
Cosmids - 40-45kb
Artificial chromosomes (eg BAC, YAC, PAC) - over 100kb

Question 26

List the 2 disadvantages of plasmid vectors

Answer 26

1) . Limits the insert size

2) . Standard transformation methods in E. coli inefficient, therefore need Ab resistance selection

Question 27

Define “competency”

Answer 27

The ability of a cell to take up extracellular “naked” DNA from its environment

Question 28

What are eg of naturally competent bacteria, and how can artificially induce competence in bacteria?

Answer 28

naturally-competent: Bacillus, haemophilus, streptococcus

To induce: treat with CaCl2, Rubidium chloride, DMSO

Question 29

What are the 5 advantages of using E. coli as the host bacteria?

Answer 29

1- genetic simplicity
2- Safe
3- Genome sequence known (expression of foreign proteins)
4- Ability to host foreign DNA
5- rapid growth rate

Question 30

How do you calculate transformation efficiency?

Answer 30

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

Question 31

List the types of Controls in cloning

Answer 31

Ligase control
Transformation control
Cell viability 
Contamination control
Linear DNA control

Question 32

Describe the principles behind blue-white screening and alpha complementation

Answer 32

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.