Lecture 28: Functional genomics; Vectors for gene cloning in E. coli

Question 1

What are the pros and cons of PCR amplification?

Answer 1

+ Complete genes can be amplified
precisely
+ PCR fragments can be cloned

• Need sequence for primer design
• PCR may introduce mutations

Question 2

What are the pros ad cons of DNA cloning?

Answer 2

+ Independent of sequence info
+ Mutations not a problem

• Restriction sites occur randomly, clones
  are random
• Rare clones may be difficult to find

Question 3

How can you clone PCR fragments?

Answer 3

Problem: Normal Taq adds ‘A’ at 3’ end,
preventing blunt end cloning

Solution: Use primers with 5’ tails
containing restriction sites. These
can be digested to creates sticky
ends with 5’ phosphates