Site Directed Mutagenesis

Question 1

What is the principle of SDM?

Answer 1

SDM is a PCR based approach that uses mutagenesis primers to synthesise new DNA sequences

Question 2

What is needed during mutagenesis primer design?

Answer 2

10-15bp flanking region each side of the mutation
Forward and reverse complimentary
Mismatch sequence central
40% GC and GC clamp

Question 3

How does SDM produce a synthesised plasmid?

Answer 3

Denature the plasmid and anneal primers containing desired mutation
DNA poly then extends the primers to produce a complete new plasmid - still nicked

Question 4

Why does it not contain methylated DNA?

Answer 4

DNA methyltranserases only found in vivo

Question 5

How are synthesised and template plasmids differentiated between?

Answer 5

Synthesised have no DNA so DpnI will degrade template DNA as it is methylation dependant

Question 6

How is the nick in synthesised plasmids repaired?

Answer 6

Transformation into XL-gold cells which are super competent E.coli

Question 7

What is competency?

Answer 7

Ability if cells to uptake exogenous DNA during transformation

Question 8

Why are super competent cells required in SDM?

Answer 8

Because amount of plasmid synthesised is very small (pg) so needs to be able to uptake

Question 9

How do you check that successful mutagenesis has occurred?

Answer 9

Miniprep and sequence the gene

Question 10

What changes the cycling parameters of the PCR reaction?

Answer 10

The type of mutagenesis

• point mutations - least
• amino acid changes
• multiple amino acid insertions or deletions - most