SDM

Question 1

Define gene mutation?

Answer 1

• permanent alteration in the DNA sequence of a gene

Question 2

Define mutagenesis

Answer 2

• process of producing genetic mutations

Question 3

What is SDM?

Answer 3

• PCR-based in-vitro process that reqs DNA template and specific mutagenesis primers to synthesise new DNA sequences w/ precise nucleotide changes

Question 4

What are the reqs for mutagenesis primer design?

Answer 4

• 10-15bp of flanking region each side of the mutation should be present
• total primer size 20-30bp
• avoid repeat or palindromic sequences
• forward and reverse primers need to be complimentary
• primers should have similar melting temperatures
• primers have a minimum of 40% GC content
• GC clamp in the last 5 nucleotides
• correct mismatch sequence is present
• mismatch sequence should be central within the primer
• multiple mismatches are possible within one primer but may decrease PCR
  success rate

Question 5

Steps of SDM

Answer 5

• plasmid prep –> insert gene into plasmid w/ target site for mutation
• temp cycling –> denature plasmid and anneal primers
• PfuTurbo DNA pol, extends and incorps mutagenic primers resulting in nicked circular strands
• add DpnI to completed PCR reaction to degrade template DNA –> newly synthesised plasmid seqs don’t have meth DNA as prod in vitro, allows differentiation
• plasmid transformation into XL-10 gold cells
• miniprep
• sequencing

Question 6

Why are XL-10 gold cells used in SDM?

Answer 6

• super-competent, req as amount of mutated plasmid synthesised is v small (pg)
• able to repair nicks present in mutated plasmids

Question 7

What is competency?

Answer 7

• ability for bacterial cells to uptake exogenous DNA during transformation and amount of DNA that can be successfully transformed
  correlated w/ competency