Mutagenesis

chemical mutagenesis

– DNA-modifying chemicals

– alkylating agents (nitrosoguanidine)

– deaminating agents (HNO₂)

– base analogues – increase mispairing

– intercalating agents – frameshifts

– produce point mutations

– mutations (usually) at random sites – no control

– works on cloned gene or genomic copies of gene (in vivo)

radiation

– UV radiation – pyrimidine dimers – point mutants

– ionizing radiation

– oxidative damage, strand breakage

– point mutants and chromosome rearrangements

– mutations at random sites – no control

– works on cloned gene or genomic copies of gene (in vivo)

transposons – mobile genetic elements

– large inserts in gene – knock-out mutations

– provides selectable marker

(usually antibiotic resistance)

– works on cloned genes or genomic copies of gene

(in vivo – in appropriate host for transposon)

– mutants not stable (if transposase enzyme present)

– insertion points not always random (hot spots)

restriction endonuclease cleavage/ligation

– can generate deletions between RE sites

(digest & ligate)

– or clone insertions into RE site

– e.g. antibiotic resistance cassette

(interposon mutagenesis)

– only works on cloned genes

– require convenient RE sites

exonuclease deletion

– generate sets of different sized deletions

– starting from restriction site

– e.g. exonuclease III/S1 nuclease technique

– deletions in 1 direction only

– or BAL 31 nuclease

– deletions in 2 directions from start site

– only works on cloned genes

– require convenient RE sites (e.g. multiple cloning site)

site-directed mutagenesis (Michael Smith)

– allows specific mutations in cloned genes

basic principle: (Fig. 7-4)

– anneal mutation-containing oligonucleotide primer

– to single-stranded plasmid template

– use DNA polymerase to synthesize second DNA strand

– from oligo primer (in vitro)

– second strand contains specific mutation

– transform mutated plasmid into E. coli

– use strain deficient for mismatch repair (mutS)

– screen for mutants – confirm by sequencing

– study mutants in vitro

– or reintroduce into genome

– homologous recombination

– use “suicide vector”

– will not grow in target organism

– e.g. bacterial plasmid in eukaryote

– only way for mutant to survive

– recombination into genome

requires:

1. cloned gene in single-stranded plasmid vector

– M13 vector or denatured double-stranded plasmid

2. oligonucleotide primer with desired mutation

– ~25 base oligo – allows primer to bind well

(even with mismatch)

– mismatch in centre of primer

– least interference with primer annealing

– no secondary structure – self-hybridization

primer may contain:

– mismatched base (point mutation)

– extra sequence (insertion mutant)

– homology to 2 sites on template

– template between sites “loops out”

– causes deletion

– can also use mix of different primers

– get mix of different mutants at same site

3. DNA polymerase – synthesizing second strand in vitro

– T7 sequenase

– good processivity

– proofreading

– no 5'-3' exonuclease

– will not displace primer from template

– proofreading thermostable DNA polymerase mixes

– for PCR mutagenesis

– Taq alone has no proofreading

– different mutants than desired one

– stops DNA synthesis partway through plasmid

– if plasmid several kb long

4. selection method for mutants

– otherwise must screen all clones by sequencing

– Kunkel method (Fig. 7-5)

– prepare template plasmid in E. coli with 2 mutations

– dut – mutant for dUTPase – cell contains dUTP

– incorporated in plasmid template DNA

– ung – mutant for uracil-DNA glycosylase

– cannot fix U in DNA

– prepare mutant strand in vitro – contains no dUTP

– transform ung⁺ strain

– template strand destroyed – glycosylase activity

– multiple apyrimidinic sites in template

– mutant strand remains

– repaired to double-stranded plasmid

– elimination of unique restriction site

– do 2 mutations at once

– 1 is desired mutation, 1 destroys unique RE site

– after second strand synthesis, transform E. coli

– grow transformation mix overnight

– miniprep culture & digest with eliminated RE

– wild type plasmids digested to linear form

– mutant plasmids have no RE site – not digested

– transform competent cells

– supercoiled circular mutant plasmid transforms cells

– linear wild type plasmid transforms poorly

Note: – works best if both mutants in same primer

– otherwise must adjust annealing conditions for 2 primers

– also lose unique restriction site – may be problem

PCR mutagenesis – use PCR primer containing mutation

– get product containing mutated sequence

Problem:

– PCR produces linear products with primers at ends

– how do you mutagenise centre of gene?

1. use inverse PCR – see PCR notes

2. megaprimer technique

– do PCR using mutagenic primer in centre of gene

– & cloning primer (with RE site) at 1 end of gene

– use PCR product as “megaprimer”

– along with cloning primer at other end of gene

– get entire gene sequence with mutant

– clone using sites at end

3. overlap extension

– generate 2 PCR products

– 1 end of each product overlaps with mutation site

(need 2 mutagenic primers)

– with at least 15 base overlap

– other ends are ends of gene (with RE sites)

– denature PCR products & mix – get annealing at overlap

– PCR amplify entire mutated gene – primers at end

– and clone