Mirco Lab Midterm Quiz: Lecture 2

Question 1

What is vitro mutagenesis? What is it used for?

Answer 1

The Purpose:

Generation of mutations in DNA sequence
* Study of structure/function relationship

• Mutagenesis of DNA regulatory regions

The Application:

• Mutagenesis of coding sequences to study protein
  function, identify enzyme active sites, evaluation of
  functional size, domain identification, epitope mapping
• Protein engineering and evolution for improvement of
  the properties (activity, solubility) of proteins used in the
  industry, design of proteins with new properties

Question 2

What are the techniques in vitro mutagenesis?

Answer 2

Oligonucleotide directed mutagenesis

• PCR-mediated oligonucleotide-directed
  mutagenesis
• Mutagenesis by assembly of oligonucleotides
  carrying mutations into double-stranded
  fragments used to replace the wild type
  sequence or for de novo synthesis
• Mutagenesis by creation of libraries of truncated
  functional elements: domains, binding sites

Question 3

What are the issues with single primer method?

Answer 3

Uses single stranded template and very low efficiency

Question 4

What are the requirements?

Answer 4

selection of the progeny from the
mutant strand or suppression of the growth of
the progeny from the non-mutant (parental)
strand

Question 5

Describe the Primer Design.

Answer 5

• at least 30 nucleotides long
• Carry the mismatched base(s) in the middle
• Form hybrid with the template that is sufficiently strong
  to allow efficient priming
• Free of palindromic, repeated or self-complementary
  sequences
• Facilitate the mutant selection i.e. by introducing
  restriction site
• The larger the mutation the lower the efficiency of
  oligonucleotide directed mutagenesis

Question 6

What is the use of the PCR directed site mutagenesis?

Answer 6

Highly efficient

• Uses double-stranded templates
• The high temperature during DNA synthesis
  increases the efficiency of reaction extension
• Convenience: availability of commercial kits
• High rate of errors: proofreading polymerases,
  sequencing of the products
• More difficult cloning
• Inefficiency of PCR for longer DNA

Question 7

Explain the creation of nested deletion series. In steps.

Answer 7

1. Cut the clone with two restriction enzymes in the MCS
2. The enzyme next to the insert creates 5’overhangs
3. The enzyme toward the vector creates 3’ overhang
4. 3’ overhang is protected from ExoIII nuclease
5. 5’ overhang can be systematically shorten with ExoIII
6. Stop the ExoIII reactions at different times
7. Blunt the DNA ends with mung bean exonuclease
8. Self-ligate the plasmids with deleted parts
9. Sequence with the same primer from the vector
   Applications: for moderately sized sequences – cDNA,
   genomic clones, good for closing larger gaps