Principles Of Cloning

Question 0

Why do we clone?

Answer 0

• reproduction if specific DNA fragments in large amounts
• stable propagation of DNA sequences
• allows DNA to be studied, manipulated and expressed

Question 1

Methods of transfer of genetic material

Answer 1

• conjugation
• transduction
• transformation

Question 2

Applications of cloning

Answer 2

• construction of genetic libraries
• production of scarce proteins
• mass production of vaccines
• diagnostics
• therapeutics
• gene transfer

Question 3

What is the restriction modification system.

Answer 3

Host DNA is methylated to protect it from cleavage by REs

Question 4

What factors are Re groups based on?

Answer 4

• composition
• enzyme cofactor requirements
• nature if target sequence
• position of DNA cleavage sites relative to target sequence

Question 5

How does DNA ligase work

Answer 5

Forms phosphodiester binds between 5’ p and 3’ OH

Question 6

What is cDNA used for?

Answer 6

• expression studies

- gene structure and function studies

Question 7

4 strategies for cloning PCR products

Answer 7

• blunt end PCR cloning
• restriction site cloning
• T/A cloning
• SDM

Question 8

What must cloning vectors contain

Answer 8

• origin if replication
• selectable marker
• multiple cloning site

Question 9

What are expression vectors

Answer 9

Allow a cloned segment if DNA to be translated into a protein inside a cell

Question 10

What must expression vectors contain?

Answer 10

• in vivo promoter
• antibiotic selection
• sequencing primers

Question 11

What is a linker.

Answer 11

Small dsDNA that contains a new RE site

Question 12

What is directional cloning?

Answer 12

Cut both vector and insert with 2 different REs

Low background of non-recombinants

Question 13

What does AP do?

Answer 13

Removes 5’ phosphatase from vectors to prevent self-ligation
Decreases non-recombinants
NB in blunt-end cloning

Question 14

How to calculate molar ratios

Answer 14

(Size of insert in kb/size of vector in kb) x ng of vector = ng of insert needed for a 1:1 ratio

Question 15

Competency

Answer 15

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

Question 16

How to increase competency

Answer 16

High ca2+ conc makes small holes in the cell membrane

Question 17

Transformation efficiency

Answer 17

Number of colonies per ml/DNA conc transformed

Question 18

Steps to isolate plasmid DNA

Answer 18

• re suspend in solution 1 (tris-HCl buffer, EDTA, Glucose)
• add solution 2 (SDS, Naoh)
• add solution 3 (potassium acetate, acetic acid)
• precipitate plasmid DNA with isopropanol
• restriction mapping

Question 19

Types of plasmid DNA

Answer 19

Uncut
- nicked
- linear
- supercoiled
Cut
- linear