Lecture 17

Question 1

What is PCR - recap yap

Answer 1

An invivro technique used to amplify up a specific region of DNA

• primers (short regions of DNA) that are designsed to amplify the specific section they are in front of

Question 2

How can we clone PCR products?

Answer 2

Restriction cut sites can be created on the ends of PCR products by adding to the end of the PCR primers

(Primers designed with a bit of extra sequence added onto them, this extra extra sequence is not going to base pair with the template DNA, instead will add on the extra sequence when the DNA is amplified up as shown in picture)
(We can design added sequence to be recognised by restriction enzyme and thus add on restriction enzyme cut sites to either side of the amplified fragment)

Question 3

Cloning PCR products - two main ways

Answer 3

• adding a restriction enzyme cut site
• TA cloning

Question 4

When cloning PCR products - how does TA cloning work?

Answer 4

Taq DNA polymerase adds on an adenine at 3’ end of PCR product
The vector has a 3’ thymine over hang
The vector can’t relegate on itself, so only clones with inserted DNA will survive

Question 5

Answer 5

Question 6

What do we do in terms of clowning if we have multiple DNA fragments that we want to join together?

Answer 6

Gibson assembly

Question 7

What does Gibson assembly do?

Answer 7

Can rapidly assemble multiple (more then 10) DNA fragments in one reaction

Question 8

Key to Gibson assembly

Answer 8

• the end of the fragment that you want to join to the next fragment must have an overlapping sequence
• all fragments are added to a Gibson assembly master mix which contains an exonuclease, DNA polymerase, DNA ligase
• exonuclease chews 5’->3’, creating a single stranded complementary region
• single stand region anneals
• gaps are filled with polymerase and ligase

Question 9

In the Gibson assemble, all linear, overlapping fragments are assembled together in a single step using a _ enzyme cocktail

Answer 9

3

Question 10

What do we do to clone a gene if a DNA sequence that doesn’t exist in nature or is difficult to isolate? Or we want to save time we can…

Answer 10

• synthetic DNA - buy our synthesised DNA in a vector
• gblocks - buy synthesised lengths of DNA blocks (up to 2Kb)

Can create restrictions sites or Gibson sites in the synthesised DNA sequence