In vitro mutagenesis

Question 1

what is forward genetics

Answer 1

start with a mutant phenotype and work towards identifying the gene whose defect is causing that mutant phenotype

Question 2

what is reverse genetics

Answer 2

start with a gene of interest, which is cloned, and works towards a mutant phenotype

Question 3

what is in vitro mutagenesis

Answer 3

Production of random or specific mutations in a segment of cloned DNA. The DNA is reintroduced into an organism to assess the effects of the mutagenesis (reverse genetics)

Question 4

why is in vitro mutagenesis important

Answer 4

More convenient as know where mutation is, controlling where mutation takes place
It detects the mutation doesn’t require long process

Question 5

what are the approaches to mutate a plasmid in vitro

Answer 5

random mutations

site-directed

Question 6

what is random mutations of a plasmid in vitro used for

Answer 6

• Helps to identify location of boundaries of a particular function of a DNA segment
• Used when a simple genetic screen (if available)
• Used as a first step when little is known about the function encoded by the DNA of interest

Question 7

what is the benefit of random mutations of plasmid in vitro

Answer 7

• Narrows the focus from a large gene to a smaller region (but does not help to explain how things work at molecular level)

Question 8

when is site-directed mutation of plasmid in vitro used

Answer 8

• Used to define the role(s) of specific sequences

Question 9

why is site-directed mutation of plasmid in vitro used

Answer 9

• It places or targets a mutation exactly where it is needed

Question 10

what does sire-directed mutation of plasmid in vitro provide

Answer 10

powerful tool for protein function analysis by allowing changes in protein structure

Question 11

what are the approaches to mutate a plasmid in vitro

Answer 11

1. Restriction endonuclease (only if the restriction site is present in the DNA segment of interest) (read palindromic sequences)
2. Linker insertion mutagenesis
3. Nested deletions
   > Unidirectional
   > Bidirectional
4. Oligonucleotide-directed mutagenesis
5. Cassette Replacement
6. PCR directed mutagenesis

Question 12

what is the general strategy for mutation plasmid in vitro

Answer 12

1. Mutate the plasmid DNA in vitro
   AmpR – ampillicin resistance
2. transform bacterial cells with mutated plasmid DNA
   Select correct ones
3. Screen for colonies that contain mutant plasmid DNA
   Test for function genetic screen or selection

Question 13

what happens in restriction endonuclease in vitro mutagenesis

Answer 13

1. Identify a restriction site in the region of interest to be mutated
2. Cut the plasmid DNA with the restriction enzyme present in the area (eg. EcoRI)
3. Manipulate the linear fragment using either of these strategies:
   > S1 nuclease removes single stranded nucleotides to leave a blunt end or (introducing a deletion)
   > DNA Polymerase + dNTPs are added to fill in the sticky ends
4. Ligate the blunt ends. The restriction site is eliminated

Question 14

what can be used to create a mutation by manipulation of a restriction site

Answer 14

S1 nuclease

DNA polymerase + dNTPs

Question 15

what is formed when mutation by manipulation of restriction site used

Answer 15

S1 nuclease plasmid has 4bp deleted

DNA polymerase + dNTPs plasmid has 4bp inserted

Question 16

what are other types of in vitro mutagenesis

Answer 16

1. Linker insertion mutagenesis (introducing a linker restriction site)
2. Nested deletions (using exonuclease III)
   > Unidirectional
   > Bidirectional

Question 17

what happens in oligonucleotide-directed mutagenesis

Answer 17

Isolate plasmid DNA from a bacteria strain with DNA methylase activity
Add mutagenic oligonucleotide primers and anneal
Extend mutagenic oligonucleotide primers
PCR, eventually will have copies, new segment of DNA will align to each other
Digest PCR products with Dpn1, which cuts only methylated GATC sites, plasmids with mutations created are left
Transform into bacteria, sequence DNA to verify that the
mutation has been created

Question 18

what does oligonucleotide-directed mutagenesis incorporate

Answer 18

a mutant oligo into one strand of plasmid DNA. The oligo is flanked by 8-12 nt of the wild-type sequence on either side

Question 19

what is the oligo sequence in oligonucleotide-directed mutagenesis like

Answer 19

sequence of the oligo is complementary to the template except for the nucleotides that define the mutation

Question 20

what happens to the strains when replicated in oligonucleotide-directed mutagenesis

Answer 20

Both strains replicate and segregate into separate mutant and wild-type plasmids

Question 21

what happens when the plasmids are introduced into cells in oligonucleotide-directed mutagenesis

Answer 21

mismatch repair system often repairs the mutated base to the complementary base in the wild-type strand before it has a chance to replicate. So the mutant plasmids are underrepresented relative to the wild-type plasmids

Question 22

how can mutated plasmids destroy wild-type template in oligonucleotide-directed mutagenesis

Answer 22

mutated plasmids can be enriched with methods that destroy the wild-type template strand of DNA by using a bacterial mutant strain that contains a degrading enzyme that attacks the wild-type DNA

Question 23

what happens in casette replacement

Answer 23

1. plasmid DNA is restricted with two different enzymes to flank the target and to remove a small wild-type sequence
2. Two synthetic oligos are ligated that contain:
   • mutant sequence
   • compatible directed ends
3. Ligate cassette with the original plasmid and transform into competent cells

Question 24

what is PCR directed mutagenesis

Answer 24

A mutation can be introduced anywhere in a PCR produced DNA fragment

Question 25

what happens in PCR directed mutagenesis

Answer 25

1. Introduce a single base mismatched between an amplification primer and a template sequence.
2. Carry out 2 PCR reactions to produce 2 overlapping DNA fragments that bear the same mutation in the overlap region.
3. The overlap in sequence allows fragments to hybridize.
4. One of the two hybrids is extended by DNA polymerase to produce a duplex fragment

Question 26

how are colonies screened to see if they are transformed with mutant plasmid DNA

Answer 26

Using radio labelled probes to detect bacterial colonies

1. Radioactively label a mutagenic probe by phosphorylating its 5’ end using 32P-ATP and T4 polynucleotide kinase
2. Replicate a plate onto nitrocellulose as done with library screen
3. Denature DNA and neutralize
4. Hybridize with a probe and wash under stringent conditions
5. Detect colonies transformed by mutant plasmid using autoradiography

Question 27

how are mutant plasmids screened using mutagenic oligonucleotides as a probe

Answer 27

mix of colonies containing mutant and wild type plasmids plated onto nitrocellulose
make replica filter
denature DNA, neutralise
add 32p, radioactive mutagenic oligonucleotide
labeled probe hybridises at room temp to mutant and wild type DNA
wash at higher temp
(probe remains bound to mutant DNA and wild-type probe dissociates)
isolate mutant DNA from colony

Question 28

what can protein engineering be used for

Answer 28

determine the role of one or several amino acids in one protein

Question 29

what are the approaches to mutate a plasmid in vitro

Answer 29

random mutations 
site directed mutagenesis
restriction endonucleases
linker insertion mutagenesis 
nested deletions
oligonucleotide-directed mutagenesis 
cassette replacement  PCR directed mutagenesis

Question 30

what happens in site specific mutagenesis using PCR

Answer 30

first section read forms PCR with primers 1 and 1M
second section read formaPCR with primers 2and 2M
the primers from these sequences are removed using exonuclease enzymes, combine sequences, denature and reanneal
3’ extension by DNA polymerase
PCR with primers 1+2