SDM

Question 1

What are the two methods for SDM?

Answer 1

Classical
PCR-based Methods

Question 2

Describe the process of Classical SDM.

Answer 2

There is a single primer binds to a ssDNA template derived from the M13 viral based vector. The primers bind to the template and the synthesis of the new strand carrying the mutation is carried out by T4 or T7 polymerase. Inserted into bacteria and plated onto agar. Selected by radioactive isotopes.

Question 3

How is an insert mutagenesis made?

Answer 3

Mutant oligonucelotide carrying a sequence to be inserted sandwiched between two regions with sequences complementary to sites on either sides of the target side in the template

Question 4

How is a deletion performed via mutagenesis?

Answer 4

Mutant oligonucleotides spanning the region to be deleted, binding to two separate sites, one on either side of the target

Question 5

What is different between PCR and SDM primers?

Answer 5

SDM primers are slightly longer in length (>20) and even longer if more than one substitution is required. Still complementary to the sequence and free of self-complementary sequences

Question 6

What type of exonuclease activity does the DNA polymerase have and why not the other one?

Answer 6

3-5’ exonuclease activity and not 5’-3’ exo because it won’t degrade primers

Question 7

What are the disadvantages of using the classical method?

Answer 7

Need ssDNA template
not very good results
non-thermostable poly
radio-labeled oligos make it more difficult

Question 8

Describe the NEB SDM process.

Answer 8

Perform DNA amplification by PCR-primer design
Remove parental DNA through DpnI (cuts methylated DNA, changed DNA isn’t methylated)
Circularization through ligation
Transformation
Plating

Question 9

What is the purpose of Random Mutagenesis and what is the general process?

Answer 9

Will change a random nucleotide with an error prone DNA polymerase with limiting the concentration of one dNTP in the presence of dITP which allows base pairing with A, C or T

Question 10

What is the general process of Gene Shuffling?

Answer 10

1. Select a set of related gene sequences
2. Treatment with DNase to generate random fragments
3. Run PCR cycles (but no primers because DNA samples are all similar and can act as their own types of primers)
4. Mutant selection for a function or phenotype

Question 10

What are some other forms of PCR that could be used for introducing the mutation?

Answer 10

Gibson Assembly and Overlap Extension

Question 11

What is the preferred DNA type for Sanger sequencing?

Answer 11

SsDNA from the M13 bacteriophage