Lecture 24: Making Mutations

Question 1

What are random mutations>

Answer 1

1. Occur spontaneously or induced by
   chemicals or radiation
2. Base changes; deletions; inversions;
   duplications; transposon insertions; etc
3. Mutations in a specific gene are rare
   (e.g., 10-6) and may be difficult to find

Question 2

What are random mutations>

Answer 2

1. Occur spontaneously or induced by
   chemicals or radiation
2. Base changes; deletions; inversions;
   duplications; transposon insertions; etc
3. Mutations in a specific gene are rare
   (e.g., 10-6) and may be difficult to find

Question 3

What do you require to generate specific mutations

Answer 3

DNA sequence information. Made possible by sequencing technologies, especially with NGS.

Question 4

How does homologous combination generate mutations

Answer 4

Take a plasmid/piece of DNA and insert it into the target gene to be mutated. To do this, parts of the sequence must be homologous, especially at the ends.

When the dsDNA is separated (into ssDNA), the plasmid may anneal (due to homologous regions) to the target gene. The DNA polymerase then has a small chance of going along the gene, then straight too and around the plasmid, then continuing. This happens at a low rate but across millions of cells.

UNSTABLE AND REVERSIBLE

Question 5

How can homologous recombination be made irreversible?

Answer 5

Using 2 cross-over sites; with a homologous region at either end of the LINEARIZED insertion DNA.

PART OF THE CHROMOSOME IS “REPLACED” WITH THE LINEAR FRAGMENT

Question 6

How and why can we make point mutations?

Answer 6

1. HR leads to complete knock-out of the
   gene, which if its essential, will just
   lead to death
2. point mutations are more subtle (bp or
   aa change)
3. Perform PCR with mutagenic primers
   which both contain a mutant
   sequence
   a. One amplifies first half of the gene
   b. other amplifies the second half