Molecular Cloning Method

Question 1

What’s a gene cloning ?

Answer 1

A process of producing individuals or group of individuals and organisms with identical DNA either naturally or artificial

Question 2

What do you need to clone a gene ?

Answer 2

1- a vector with Ori and MCS marker
2- insert with a proper sticky ends
3- restriction enzyme
4- DNA Ligase

Question 3

Restriction Enzymes

Answer 3

These enzymes prevent invasion by foreign DNA such as viral DNA, by cutting it up, they cut at sites within the foreign DNA rather than chewing it hens the name “ restriction endonuclease “ and not exonuclease

Question 4

Heteroschizomers

Answer 4

Enzymes that recognizes different sites in identical sequences ( eg. Smal and Xmal )

Question 5

Isoschizomers

Answer 5

Enzyme that cuts at the same site in the same sequence

Question 6

What are the advantages of Restriction Enzymes ?

Answer 6

• ability to cut DNA strands reproducibly in the same places
• make staggered cuts in two DNA strands
• creates sticky ends that makes it easier to stick two different DNA molecules together

Question 7

Palindromes

Answer 7

Sequence with twofold symmetry , read the same forward and backwards

Question 8

Restriction enzymes if they can cut up invading viral DNA, why do they not destroy the host cells own DNA ?

Answer 8

Almost are restriction enzymes are paired with me Methylases, that recognize and methylate the same DNA sites, after methylation DNA sites are protected against most restriction Endonuclease

Question 9

R-M system is ?

Answer 9

Restriction endonuclease and methylase are collectively called the restriction-modification system

Question 10

whats a vector ?

Answer 10

vectors function as DNA carriers to allow replication of recombinant DNAs

Question 10

whats a vector ?

Answer 10

vectors function as DNA carriers to allow replication of recombinant DNAs

Question 11

whats a vector ?

Answer 11

vectors function as DNA carriers to allow replication of recombinant DNAs

Question 11

whats a vector ?

Answer 11

vectors function as DNA carriers to allow replication of recombinant DNAs

Question 12

importance of vectors to foreign DNA

Answer 12

The foreign DNA has no origin of replication a site where the DNA replication begins, It cannot replicate unless it is placed in a vector that does have an origin of replication.

Question 13

gene cloning experiments depends on two things…

Answer 13

a vector and a piece of foreign DNA that depends on it vector

Question 14

how does the recombinant DNA is being introduced into bacterial cells ?

Answer 14

by transformation, the two ways to do this is ;
1- incubating the cell in a concentrated calcium salt - makes the membrane leaky - allows the DNA entrance to the leaky cells
2- also by using high voltage to drive DNA into the cell, process called electroporation