Use of Molecular Approaches to Clone DNA (Recombinant DNA Technology)

Question 1

What is the two pathway of amplication

Answer 1

In vivo amplification - insert a piece of DNA into a vector which then insert it into a bacteria host. host multiply will also multiply the gene of interest
In vitro amp- PCR using primer and ssDNA oligonucleotides

Question 2

Requirement for in vivo DNA amp

Answer 2

-need to cut the vector at a particular site and rejoin them again: molecular toolkit
- require a suitable vector: plasmid, phage

Question 3

What is restriction

Answer 3

-A method which some bacteria use to defend against phage
-involve in cutting and degration of phage DNA
-Done with restriction endonuclease

Question 4

What is modification

Answer 4

a method phage use to overcome restriction mechanism
involve chemically modifying the phage by adding methylase (add methyl group)

Question 5

Type 2 restriction system

Answer 5

-endonuclease and methylase are found in different gene
-endonuclease regconise a specific DNA sequences know as restriction site
-Restriction site have two fold rotational symmetry

Question 6

Two way of DNA cutting

Answer 6

-Blunt cut which create blunt end
-stagger cut which create staggering end (3’ 5’ overhang)

Question 7

probability of a random piece of enzyme having the same restriction site

Answer 7

-chance of each nucleotide of the chain times each other

Question 8

Annealing follow restriction

Answer 8

• single strand overhang can anneal with each other
  -thought intramolecular association (same gene) is more frequent than intermolecular association
  -can occur between DNA of different origin

Question 9

joinning DNA ligation

Answer 9

-anneling involves non-covalent hydrogen bond between complementary bases (unstable)
-DNA ligase is use to seal the nick between the backbone of the two annealed DNA strand (stabalise)
ーcan repair nick in any DNA but need a cofactor (ATP)

Question 10

ligate compatible end

Answer 10

-The same end (complimentary) can be joined toghter with DNA ligase
-Any blunt end can join with each other with DNA ligase but inefficient

Question 11

step in generating recombinant DNA

Answer 11

1. Cleve donor and vector DNA with endonuclease
2. mix and ligate DNA molecule together

Question 12

Key consideration for gene cloning

Answer 12

1/ need to introduce plasmid into bacteria (transformation)
2/ Need to identify transformed bacteria- (select for the presence of the plasmid)
3/ Need to identify transformed bacteria with recombinant plasmid- select for the presence of a DNA in the plasmid
4/ Need plasmid that are design for the task (genetic engineering)

Question 13

Identification of transformed cells

Answer 13

-Transformation is an inefficient process as only 0.01% will enter the plasmid
-there is a need to distinguish between plasmids
-this can be done by selective marker (color or resistance in the vector)

Question 14

Visual screening for insertional inactivation

Answer 14

-screen to identify bacteria with recombinant plasmids is based on loss of gene activity (because a huge chunk has been inserted in the gene)

Question 15

Blue white screening

Answer 15

-insertional inactivation of lacZ gene
-IPTG as inducer for lacZ (constitutional activity)
-X-gal as an substrate for B-galactosidase (blue =B galactosidase activity)
-look for white colony

Question 16

Characteristic of plasmid for clonning

Answer 16

-low molecular weight (3kb)
-origin of replication
-unique restriction site
-selectable marker
-insertional inactivation that does not disrupt the reading frame