Test 4- Lecture 11: Molecular Cloning And Recombinant DNA

Question 1

Recombinant DNA technology

Answer 1

Allows scientists to manipulate DNA fragments in order to study them in the lab

Question 2

What does recombinant DNA technology involve?

Answer 2

Using a variety of lab methods to put a piece of DNA into a bacterial or yeast cell
Once in, the bacteria or yeast will copy the DNA along with its own

Question 3

What has recombinant DNA technology been successfully applied to?

Answer 3

Make important proteins used in the treatment of human diseases, such as insulin and growth hormone

Question 4

What kind of tool is recombinant DNA technology?

Answer 4

An extremely important research tool in biologi

Question 5

Recombinant DNA technology uses what and why?
Why not the other kind?

Answer 5

Plasmid DNA; can cut and manipulate
Chromosomal DNA; can do but too complicated

Question 6

Scientists prefer to handle what kind of DNA in recombinant DNA technology?

Answer 6

Small DNA (Plasmid DNA)

Question 7

If you want to clone or insert DNA of interest, what to do?

Answer 7

Can cut & paste and put back in the bacteria
When bacteria multiply, plasmid DNA also multiply

Question 8

What was the main technology before PCR?

Answer 8

Recombinant DNA technology

Question 9

Recombinant DNA technology diagram
(right to left, top to bottom)

Answer 9

DNA of interest
DNA of interest and plasmid digested with restriction enzyme
Bacterial cell

Plasmid
DNA ligase
Recombinant DNA
Recombinant DNA introduced into bacterial cells
Recombinant DNA copied and cells divide

Question 10

Molecular cloning refers to (3)

Answer 10

Isolation of a DNA sequence from any species (often a gene)
Its insertion into a vector for propogation, without alteration of the original DNA sequence

Question 11

Two aspects of molecular cloning have revolutionized a scientist’s ability to manipulate DNA:

Answer 11

1. The ability to cut, alter, and join DNA molecules in vitro
2. The ability to copy and mass produce recombinant DNA molecules in host/vector systems

Question 12

Advantage of putting DNA sequence in plasmid

Answer 12

When the DNA is replicated, there is no mistake in the replication process because bacteria has mechanism by which it can correct the mistake

(No mutation you can induce during multiplication/propagation of DNA

Question 13

What is the host of the vector?

Answer 13

The bacteria

Question 14

Recombinant DNA molecules

Answer 14

The DNA which you cut and paste
Plasmid DNA insert is recombinant DNA

Question 15

The basic cloning workflow includes 4 steps

Answer 15

1. Isolation of target DNA fragments (inserts)
2. Ligation of inserts into an appropriate cloning vector, creating recombinant molecules (plasmids)
3. Transformation of recombinant plasmids into bacteria or other suitable host for propagation
4. Screening/selection of hosts containing the intended recombinant plasmid

Question 16

Ligation of DNA inserts into an appropriate cloning vector (2)

Answer 16

Ligation: joining of those DNA pieces; put insert into tiny plasmid DNA
Cloning vector: plasmid DNA

Question 17

Transformation of recombinant plasmids into bacteria (2)

Answer 17

Recombinant plasmids: recombinant DNA molecule, red and white DNA fragments (in video)
Do ligation reaction (cut and paste in test tube, not in bacterial cell- isolated and purified way)

Question 18

Transformation…

Answer 18

Is not 100%
Always get a mixture of untransfected and transfected cells