in vitro cloning

Question 1

What is in - vitro cloning?

Answer 1

• DNA fragments can be amplified using in vitro cloning where copies are made outside a living organism
• the polymerase chain reaction is automated, rapid and efficient

Question 2

What is the process of in - vitro cloning?

Answer 2

1. The DNA fragment is copied
2. DNA polymerase is used which joins nucleotides
3. Primers - short sequences of DNA nucleotides that have a set number of bases complementary to the bases at the end of DNA fragments

Question 3

What are the stages of in - vitro cloning?

Answer 3

1. Seperation of the DNA strand
2. Addition of primers
3. Synthesis of DNA

Question 4

What is stage one?

Answer 4

The DNA fragments, primers and DNA polymerase are placed in a thermocycler at 95 degrees causing the hydrogen bonds in the DNA fragment to break, seperating the two DNA fragments.

Question 5

What is stage two?

Answer 5

The mixture is cooled to 55 degrees, causing primers to join their complementary base pairs
- these primers provide a starting point for DNA polymerase to bind to the new DNA strands

Question 6

What is stage three?

Answer 6

The temperature is increased again to 72 degrees and DNA polymerase begins joining the free DNA complemtary bases to the DNA strands, starting at the primers
This produces 2 double stranded DNA strands
The cycle then repeats

Question 7

How much DNA is produced?

Answer 7

Each PCR cycle produces x 2 more DNA fragments
SO the first produces 4 strands
The second produces 8 new strands

Question 8

What are the advantages of in vitro replication?

Answer 8

• rapid = the polymerase chain reaction is fast
• doesnt require living organisms

Question 9

What are the advantages of in vivo replication?

Answer 9

• accurate
• useful for inserting gene into another organism
• no risk of contamination = use of the same restriction endonuclease
• cuts specific genes
• transformed bacteria produces large quantities of the gene products