Topics: In Vitro Mutagenesis

Applications:

1. Determine the functional groups of a protein

2. Protein engineering- Enhance or modify enzymatic or protein function "Directed Evolution

Improve proteins with single amino acid changes - pharmocokinetics, structure stability bioavailability

Ex.- engineer insulin that are monomeric, absorb 2-3x efficiently

Ex. TPA- engineered to be resistant to oxidative stress

Ex.- improve enzymes- pH, oxidative, thermal and alkaline stability, substrate specificity,

1. Mutagenize plasmid DNA in vitro

2. Transform mutagenized plasmid DNA into competent cells

3. Screen for colonies that contain mutant plasmid DNA

1. Plasmid DNA is cut with a restriction enzyme (Eco RI is used in the example)
2. Linear fragment is manipulated

1. Plasmid DNA is treated with low concentrations of DNAase I in the presence of Mn
2. Under these conditions, the enzyme makes ds cuts randomly
3. Random cut linear DNA ligated with linkers containing a restriction enzyme site (Eco RI linkers)
4. Linear DNA + linkers is restricted to create sticky ends on linkers
5. Ligate to circularize plasmid DNA and transform into competent cells

Transposon Mutagenesis- Described Dr. Mike Cleary's lecture with Tn5 to screen for mutated plasmid

1. Linearize plasmid clone by restriction enzyme digestion at the 5' side of gene (upstream)
2. Treat with Exonuclease which digest DNA from both sides of molecule
3. Remove sample and stop reaction at different time points
4. Ligate with an Eco RI linker, digest with Eco RI to create sticky ends and ligate into a new vector that has compatible sticky ends. Transform into competent cells

2. After DNA replication of this heteroduplex plasmid, any progeny plasmids that arise by replication of the wild-type strand will be homozygous for the wild-type allele, and any plasmids that arise by replication of the mutant strand will be homozygous for the mutant allele.

3. The resulting population of plasmids contains both wild-type and mutant plasmids that may only differ by a single base-pair mutation in a sequence which lacks an easily assayed phenotype.

4. Furthermore, when introduced into cells the 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.

Improved methods of in vitro mutagenesis destroy the wild-type template strand of DNA and thus favor replication of the mutant strand

DNA template is grown in a dut ung mutant of E. coli.

The dut gene encodes dUTPase which degrades dUTP. (Therefore dUTP accumulates in dut strains & U sometimes take the place T during DNA replication).

The ung gene encodes uracil N-glycosylase which normally removes U from DNA.

dut ung mutant - U is occasionally incorporated into DNA and this error is not repaired- not mutagenic.

A mutagenic oligonucleotide is annealed to a ssDNA template

Add DNA polymerase, dNTPs but no dUTP,

Add Ligase to circularize - template has Us Mutant has T's

Transformed into an ung+ recipient cell.

The uracil N-glycosylase excises the U leaving apyrimidinic (AP) sites in the template strand.

Presence of AP sites makes the DNA strand biologically inactive because it cannot be replicated by DNA polymerase

The plasmid DNA is cut at the AP sites by specific endonucleases. Hence, when the dsDNA is introduced into the ung+ recipient , only the mutant strand will be replicated.

Note that the dut ung method provides an enrichment for the mutant plasmids, not a selection -- typically only about 50% of the resulting plasmids carry the mutation introduced on the oligonucleotide.

1. Use two mutant oligos to amplify mutation
2. Digest product with Dpn I- a restriction enzyme which specifically cleaves fully methylated DNA template DNA but not the DNA made in vitro.
3. Transform into host cells that will repair nicked DNA with the mutation.

Variation by Sawona et al

1. Use one or more oligos containing mutation that have been 5' Phosphorylated by T4 polynucleotide kinase
2. Amplify target via PCR in the presence of a highly processive, high fidelity thermophillic DNA polymerase (such as Stratagene's pfu) and taq T4 ligase to create circular products
3. Digest product with DpnI- to nick template DNA
4. Transform into host cells that will repair nicked DNA with the mutation

1. Plasmid DNA restricted with two different enzymes to flank the target and remove a small wild-type sequence
2. Two synthetic oligos are ligated to contain:

* Primers should be between 25 and 45 bases in length,

* (Tm) of the primers should be greater than or equal to 78 C.

For calculating Tm for primers intended to introduce insertions or deletions, N=primer length % are whole numbers

Tm = 81.5 + 0.41(%GC) -675/N - %mismatch

* The desired mutation (deletion or insertion) should be in the middle of the primer ~10-15 bases of correct sequence on both sides.

* Minimum GC content of 40% and should terminate in one or more C or G bases.

* Must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.

* It is important to keep primer concentration in excess. Varying the amount of template while keeping the concentration of the primer constantly in excess.

• Be free of palindromic, reiterated or self-complemenatry sequences that might form stable structures and prevent priming

1. Radioactively label a mutagenic probe by phosphoylating its 5' end using P32- ATP with T4 polynucleotide kinase
2. Replica plate onto nitrocellulose as done with library screen
3. Denature DNA; neutralize
4. Hybridize with probe and wash under stringent conditions
5. Detect colonies transformed by mutant plasmid by autoradiography

Based on the observation that PCRs require the 3'terminal base of the oligo to be perfectly complementary to the DNA template.

Glisic & Alavantic (1996) Four primers required used to amplify target DNA under standard PCR conditions, products analyzed with agarose gel-electrophoresis. No need to mini-prep DNA, toothpick labeled colonies on plate in 20 µl TE, vortex and take 2 µl for PCR reaction.

Primer 1- forward- to amplify target region (+ control)

Primer 2- allele specific forward- test for mutant (experimental) Use for 2^nd round of amplification

Primer 3- allele specific forward- test for wild-type Use for 2^nd round of amplification

Primer 4- reverse to amplify target region (+ control) See Figure

2. Denature PCR product (heat or formamide, or NaOH) to make single stranded. Single stranded DNA duplex fold into complex 3D structures. Differnet sequences can affect mobility in a gel.

3. Electrophoresis of single stranded DNA through a gel at neutral pH- detect differences between mutant and wild-type strands of DNA.

Amersham- Sculptor IVM Mutagenesis kit & Unique Site Elimination (USE) elimination kit

BioRad- Muta-gene in vitro mut. Kit

CLONTECH- Transformer sitedirected mutageneis kit

Promega- Altersite II; Gene Editor; Interchange; Error Prone PCR

Stratagene- Quik Change; ExSite PCR-based; Chameleon DS site-directed mutagenesis kit

NEB- Code20kit

Primrose, Twyman & Old 6^th Edition Principles of Gene Manipulation

Recombinant DNA 2nd Edition Watson, Gilman, Witkowski Zoller