15 - Molecular Cloning

Question 1

Uses of DNA cloning

Answer 1

• Gene mapping
• DNA sequencing
• Identification of unknown genes
• Allows manipulation of DNA

Question 2

Process of cloning gene

Answer 2

• Choose strategy for isolating gene of interest
• Choose a destination (e.g. plasmid)
• Choose destination host (e.g E. coli)
• Choose method for identifying recombinant E. coli carrying gene of interest

Question 3

Two basic strategies for isolating gene from genomic DNA

Answer 3

• Cloning random restricted DNA fragments (library of randomly cloned fragment)
• Cloning specific PCR fragment (single cloned fragment)

Question 4

Advantages of cloning randomly restricted fragments

Answer 4

Do not need to know the sequence of the target gene

Question 5

Disadvantages of cloning randomly restricted fragments

Answer 5

• Not specific for gene of interest
• RE may cut inside gene of interest

Question 6

Advantages of cloning specific PCR fragment

Answer 6

Very specific amplification of gene

Question 7

Disadvantages of cloning specific PCR fragment

Answer 7

Must know sequence of target gene for design of primers

Question 8

Tools required for cloning random DNA fragments

Answer 8

• High molecular weight DNA
• Restriction enzymes
• T4 DNA ligase
• Bacterial transformation

Question 9

Restriction enzymes

Answer 9

Enzymes that can cleave the
phosphodiester bond at specific sites in the DNA duplex

Question 10

Properties of restriction enzymes

Answer 10

• Named according to source
• Require Mg++ for activity
• Cut at fixed position within restriction site (sticky or blunt ends)

Question 11

Restriction site

Answer 11

4-10 base pair palindrome where restriction enzymes cut

Question 12

Frequency of cutting of restriction enzymes

Answer 12

Related to the length of the recognition sequence

Question 13

Why is buffer of pH 8 important

Answer 13

To ensure DNA remains negatively charged

Question 14

Which node does DNA migrate to in electrophoresis

Answer 14

Anode (positive)

Question 15

What do plasmid cloning vectors contain

Answer 15

• Selectable marker
• Multiple cloning site
• Origin of replication

Question 16

Mechanism for detecting recombinant vectors

Answer 16

• Insertional inactivation (blue white screening)
• Gain of function (phenotype e.g antibiotic resistance)

Question 17

Ligation

Answer 17

Creation of a phosphodiester bond between the 3’ hydroxyl of one nucleotide and the 5’ phosphate of another

Question 18

Requirements for ligation

Answer 18

• DNA compatible ends
• T4 DNA ligase from T4 phage
• ATP
• Mg++
• Optimal buffer pH
• Incubation at 16ºC

Question 19

Mole ratio of vector to insert for sticky ligation

Answer 19

1:3

Question 20

Mole ratio of vector to insert for blunt ligation

Answer 20

1:1

Question 21

Compatible ends

Answer 21

• All blunt ends
• Sticky ends that are identical

Question 22

How can sticky ends be made into blunt ends

Answer 22

Fill in 5’ overhangs or removal of 3’ overhangs by klenow fragment or T4 DNA polymerase

Question 23

Halting of self ligation of vector

Answer 23

• Alkaline phosphatase removes 5’ phosphates from termini of cut DNA
• Must be removed before ligation

Question 23

Sources of alkaline phosphatase

Answer 23

• Bacterial
• Calf intestinal
• Shrimp

Question 24

Expression of cloned gene in new bacterial host

Answer 24

- DNA ligation (formation of clones) - Transformation into E. coli - Bacteria plated on medium + antibiotic - Overnight incubation - Screen for gene expression

Question 25

Isolation of plasmid

Answer 25

- Screen phenotype (e.g haemolysis) - Pick colony - Grow pure culture - Purify the plasmid (Alkaline lysis method/column purification) - Analyse plasmid (restriction map/sequence)

Question 26

Steps of blue white screening

Answer 26

1. Digest foreign DNA and pUC18 with Sall 2. Ligate digested DNA together 3. Transform into E. coli host 4. Plate onto agar plates containing ampicillin 5. White colonies contain recombinant vector, blue contain self ligated vector

Question 27

β-galactosidase LacZ

Answer 27

- Cleaves lactose to galactose and glucose - Cleaves X-gal which forms blue precipitate

Question 28

Promotor P-lac

Answer 28

- Binds repressor Lacl - Transcription is induced by allolactose

Question 29

Blue white screening

Answer 29

- LacZ can be cleaved into two fragment peptides - LacZΩ and LacZα are BOTH ENZYMATICALLY INACTIVE by themselves - When co expressed, the peptides bind to each other to restore β-galactosidase activity - Both genes are under the control of LacI and transcription is induced using isopropyl-β-thiogalactoside

Question 30

E. coli host strain encodes only LacZΩ

Answer 30

- In its genome or on a plasmid - Not resistant to antibiotics unless it gets cloning plasmid - LacZ is not functional and no colony is formed

Question 31

Empty vector used for cloning encodes LacZ⍺

Answer 31

- The multiple cloning site is inside the gene encoding LacZ⍺ - When empty vector is transformed into E. coli host strain, complementation occurs - LacZ is functional and blue colony is formed

Question 32

Recombinant vector does not produce LacZ⍺

Answer 32

- The insert integrates into the multiple cloning site - Gene encoding LacZ⍺ is disrupted - LacZ if not functional, white colony formed

Question 33

Overall process of cloning

Answer 33

- Obtain vector - Obtain insert (PCR or fragmentation) - Restriction digest of vector and insert - Ligate insert and vector (T4 DNA ligase) - Transform ligation mixture in E. coli - Selection (blue white screening) - Verification (sequencing)

Question 34

Methods of transformation (vector into host)

Answer 34

- Heat shock - Electroporation