Cloning Vectors

Question 1

The most useful vectors have what three properties?

Answer 1

1. The vector DNA can be introduced into the host cell relatively easily.
2. The vector and any DNA is contains can be replicated inside the host cell.
3. Cells containing the vector can be identified in a straightforward manner, most conveniently through a novel phenotype conferred on the host by DNA sequences present in the vector (such as antibiotic resistance)

Question 2

Name some vectors and their insert sizes. [5]

Answer 2

1. Plasmid: 5-10 kb
2. Bacteriophage vector: 50 kb
3. Cosmid vector: 40-45 kb
4. Bacterial artificial chromosome (BAC): 100-200 kb
5. Yeast artificial chromosome (YAC): >1 Mb

Question 3

What’s the difference between a stringent and a relaxed plasmid?

Answer 3

• A stringent plasmid only replicated when the genome is replicated, and therefore there is a low copy number.
• Relaxed plasmids replicate autonomously, independent of the genome. Therefore there is a high copy number.

Question 4

Give an example of a plasmid vector.

Answer 4

pUC19

It has an oriV and two restriction markers for positive/negative selection by insertional inactivation

• Ampicillin resistance gene
• LacZ for blue/white selection in the presence of Xgal-containing plates (no insert is blue, insert turns plaques white)

High copy number (1,000/cell) and several multiple cloning sites and restricted use to E. coli.

Question 5

What is a shuttle vector?

Answer 5

A shuttle vector is a vector constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use.

Question 6

What are the 7 main steps in cloning a cDNA library?

Answer 6

1. Isolation and purification of mRNA
2. Synthesis of cDNA copy
3. Vector linearisation
4. Ligation
5. Transformation/transfection
6. Library screening
7. Clone characterisation

Question 7

What are the 5 main steps in cloning a DNA library?

Answer 7

1. Target DNA fragmentation
2. Vector linearisation
3. Ligation
4. Transformation/transfection
5. Library screening
6. Clone characterisation

Question 8

How is a cDNA copy made? [5]

Answer 8

1. Affinity chromatography using an oligo-dT matrix (detects the poly-A tails on mRNAs)
2. Strand synthesis using reverse transcriptase to make a DNA/RNA hybrid
3. RNase H removes the RNA
4. dsDNA is formed using polymerase
5. Manipulation of dsDNA ends is done to facilitate insertion into vectors, e.g. adaptors, linkers

Question 9

Outline the advantages and the disadvantages of creating genomic and cDNA libraries.

Answer 9

Question 10

What is a restriction enzyme? Give an example.

Answer 10

A nuclease that cleaves duplex DNA wherever the DNA molecule contains a particular short sequence of nucleotides matching the restriction site of the enzyme. Most restriction sites are palindromic.

For example, EcoRI cuts CTTAAG between the G and A. The breaks usually create sticky ends with overhangs, which are complimentary to fragments cut by the same restriction enzyme.

Question 11

What methods increase the efficiency of transformation?

Answer 11

Transformation is rare, therefore there are some methods which can be used in order to improve the likelihood of transformation:

• Polycations- highly charged molecules that interact with the negative DNA backbone and promotes attraction to the cell membrane, e.g. DEAE-cellulose, DEAE-dextran, causing DNA to enter the cell.
• Heat shock- 42 ºC causes cells to become porous, meaning DNA can enter easily.
• Protoplast formation- enzymes, such as lysozyme, degrade the cell wall. DNA enters and the cell wall reforms.
• Electroporation- shock the bacteria with high voltage (10-20 kv/cm) causing the cell membrane to become porous, allowing DNA to enter.
• Transformation- using CaCl2, causes the cell membrane to become porous so DNA can enter the cell.

Question 12

Outline bacterial artificial chromosomes.

Answer 12

BAC (bacterial artificial chromosome) vectors are artificial chromosomes based on F plasmids.

• Can take up to 350 kb inserts.
• They have origins of replication and antibiotic resistance genes.
• lacZ gene.

Question 13

Outline yeast vectors. What two types are there?

Answer 13

Yeast vectors, such as S. cerevisiae, are useful because they are eukaryotes, non-pathogenic, provide eukaryote gene expression, can be grown in high numbers, can secrete proteins and provides glycosylation. Secretion genes can be for auxotrophs such as Leu, Trp, and His mutants. Yeast are electroporated with vectors carrying these markers, hence yeast that receive the plasmid can grow on media with the amino acid deficiency. Two types of yeast vectors:

1. Plasmid derived vector: 50-100 copies per genome, constructed by fusion of plasmid with bacterial sequence, and incorporation of yeast selectable markers, such as the Leu2 gene.
2. YAC (yeast artificial chromosome) vector: can clone large DNA fragments, up to 1 Mb, and are based on yeast elements fused with bacterial sequences. After making the library, DNA is transferred directly into yeast.

Question 14

Outline bacteriophage lambda.

Answer 14

Bacteriophage λ vectors are used to take larger pieces of DNA than plasmids, and are ideal for cDNA libraries.

• They have the capacity of 10-23 kb.
• The vector has two halves which are divided up by stuffer (replaceable) DNA, which is replaced by the target DNA insert through recombination. The stuffer DNA can be the lacZ gene so that inserts can be identified with a colour change (no insert is blue, insert turns plaques white).
• Foreign DNA is amplified by infection of a bacterial lawn, then isolation of a plaque that is used to infect new bacteria. Clones are grown in suspension and the phage DNA is isolated when the cells lyse.