8 Approaches to Random MutagenesisNick OswaldTech Tips

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Random mutagenesis is an incredibly powerful tool for altering the properties of enzymes. Imagine, for example, you were studying a G-protein coupled receptor (GPCR) and wanted to create a temperature-sensitive version of the receptor or one that was activated by a different ligand than the wild-type. How could you do this?

Firstly, you would clone the gene encoding the receptor, then randomly introduce mutations into the gene sequence to create a “library” containing thousands of versions of the gene. Each version (or “variant”) of the gene in the library would contain different mutations and so encode receptors with slightly altered amino acid sequences giving them slightly different enzymatic properties than the wild-type.

Next, you could transform the library into a strain where the receptor would be expressed and apply a high throughput screen to pick out variants in the library that have the properties you are looking for. Using a high throughput screen for GPCR activity (see here for examples) you could pick out the variants from the library that were temperature-sensitive or were activated by different ligands.

Sound easy? Well, of course it’s not that easy. Creating a random mutant library that contains enough variants to give you a good chance of obtaining the altered enzyme you desire is a challenge in itself. There are many ways to create random mutant libraries, each with it’s own pros and cons. Here are some of them:

1. Error-prone PCR. This approach uses a “sloppy” version of PCR, in which the polymerase has a fairly high error rate (up to 2%), to amplify the wild-type sequence. The PCR can be made error-prone in various ways including increasing the MgCl2 in the reaction, adding MnCl2 or using unequal concentrations of each nucleotide. Here is a

good review of error prone PCR techiques and theory. After amplification, the library of mutant coding sequences must be cloned into a suitable plasmid. The drawback of this approach is that size of the library is limited by the efficiency of the cloning step. Although point mutations are the most common types of mutation in error prone PCR, deletions and frameshift mutations are also possible. There are a number of commercial error-prone PCR kits available, including those from Stratagene and Clontech.

2. Rolling circle error-prone PCR is a variant of error-prone PCR in which wild-type sequence is first cloned into a plasmid, then the whole plasmid is amplified under error-prone conditions. This eliminates the ligation step that limits library size in conventional error-prone PCR but of course the amplification of the whole plasmid is less efficient than amplifying the coding sequence alone. More details can be found here.

3. Mutator strains. In this approach the wild-type sequence is cloned into a plasmid and transformed into a mutator strain, such as Stratagene’s XL1-Red. XL1-red is an E.coli strain whose deficiency in three of the primary DNA repair pathways (mutS, mutD and mutT) causes it to make errors during replicate of it’s DNA, including the cloned plasmid. As a result each copy of the plasmid replicated in this strain has the potential to be different from the wild-type. One advantage of mutator strains is that a wide variety of mutations can be incorporated including substitutions, deletions and frame-shifts. The drawback with this method is that the strain becomes progressively sick as it accumulates more and more mutations in it’s own genome so several steps of growth, plasmid isolation, transformation and re-growth are normally required to obtain a meaningful library.

4. Temporary mutator strains. Temporary mutator strains can be built by over-expressing a mutator allele such as mutD5 (a dominant negative version of mutD) which limits the cell’s ability to repair DNA lesions. By expressing mutD5 from an inducible promoter it is possible to allow the cells to cycle between mutagenic (mutD5 expression on) and normal (mutD5 expression off) periods of growth. The periods of normal growth allow the cells to recover from the mutagenesis, which allows these strains to grow for longer than conventional mutator strains.

If a plasmid with a temperature-sensitive origin of replication is used, the mutagenic plasmid can easily be removed restore normal DNA repair, allowing the mutants to be grown up for analysis/screening. An example of the construction and use of such a strain can be found here. As far as I am aware there are no commercially available temporary mutator strains.

5. Insertion mutagenesis. Finnzymes have a kit that uses a transposon-based system to randomly insert a 15-base pair sequence throughout a sequence of interest, be it an isolated insert or plasmid. This inserts 5 codons into the sequence, allowing any gene with an insertion to be expressed (i.e. no frame-shifts or stop codons are cause). Since the insertion is random, each copy of the sequence will have different insertions, thus creating a library.

6. Ethyl methanesulfonate (EMS) is a chemical mutagen. EMS aklylates guanidine residues, causing them to be incorrectly copied during DNA replication. Since EMS directly chemically modifies DNA, EMS mutagenesis can be carried out either in vivo (i.e. whole-cell mutagenesis) or in vitro. An example of in vitro mutagenesis with EMS in which a PCR-amplified gene was subjected to reaction with EMS before being ligated into a plasmid and transformed can be found here.

7. Nitrous acid is another chemical mutagen. It acts by de-aminating adenine and cytosine residues (although other mechanisms are discussed here) causing transversion point mutations (A/T to G/C and vice versa). An example of a study using nitrosoguanidine mutagenesis can be found here.

Note: I have only mentioned two chemical mutagens but there are many others. Hirokazu Inoue has written an excellent article describing some of them and their use in mutagenesis, see here (pdf).

Another note: Chemical mutagens are, of course… mutagens and therefore should be handled with great care. Be especially careful with EMS as it is volatile at room temperature. Read the MSDS and do a proper risk assessment before carrying out these experiments.

8. DNA Shuffling is a very powerful method in which members of a library (i.e. copies of same gene each with different types of mutation) are randomly shuffled. This is done by randomly digesting the library with DNAseI then randomly re-joining the fragments using self-priming PCR. Shuffling can be applied to libraries produced by any of the above method and allows the effects of different combinations of mutations to be tested. For more information see here and here (see page 13).

In Vitro Mutagenesis:Natural mutation, in general, is spontaneous.  Any

heritable change is considered as mutation.  It can be spontaneous or induced.  Mutation cannot be predicted in nature.  Mutation at chromosomal level can be numerical (ploidy) or it can be structural (aberrations).  At molecular level mutation can be deletion of a sequence of nucleotide or nucleotides, or addition of nucleotide or nucleotides, translocation of DNA segments with in the chromosome or between chromosomes or it can be due to inversion of DNA segments. 

Point mutations are generally restricted to changes in a single nucleotide i.e. substitution of a nucleotide or addition of nucleotide or loss of a single nucleotide.

Mutation can lead to gain of a function called Gain mutation, or loss of a function called null mutation.  Mutation can conditional, where the effect of mutation is expressed or manifest only under certain condition.  Many mutations are spontaneously reversed called reverse mutations, but some mutations are suppressed called suppressor mutations.

The rate of mutations varies form one organism to another, which depends upon the phylogenetic and hierarchical position of the organism.  In that sense viruses show greater propensity for changes than Bacterial systems.  And bacterial systems are prone to mutations than higher organisms.  Haploids suffer more than diploids due to mutation of one or the other kind. 

All natural mutations are unpredictable and spontaneous and it is the primary force of nature that caused variations and those variations survived the test of the time are fixed and furthered, thus species originated in nature.  Today molecular technology that is available at hand can be used create desirable mutation under in vitro condition.  It is not just creating random mutations; it is now possible to create mutations to create new codons, new messages and new characters.  This is possible to target a single nucleotide or a group of nucleotides.  In this process one can change a single codon or add one or more codons and delete one or more codons.  Such changes in coding sequence of a particular gene can generate a protein with new conformation or new function or both; in essential it amounts to protein engineering.

Protein engineering is an emerging technology of great importance in medicine and industry.  By this

technology one can enable the protein to be stable at higher temperature and still active in non-aqueous conditions.  One can make the protein to change its affinity to the substrate and increase the activity by several folds at low Km. It is also possible to change its specificity of the substrate and function.  This is going to be one of the futuristic aspects of biotechnology.

Application of Site Directed Mutagenesis:        Proteins stable and functional at high temperatures.

        Proteins stable and functional at changed pH.

        Proteins active in non-aqueous solvents.

        Proteins don’t require cofactors for their function.

        Proteins resistance to proteases.

        Proteins with changed allosteric features.

        Proteins with changed active site and increased specific activity.

        Proteins with changed Km and Vmax with increased catalytic efficiency.

A list of few engineered proteins:       Alpha Amylase- used in bear making.

       Subtisilin-biodetergent.

       Amino cyclase- Preparation of L-amino acids.

       Bromelain- Meat tenderizer and juice clarifier.

       Catalase- anti oxidant in food preparation.

       Ficin- meat tenderizer.

       Cellulase- alcohol and glucose production.

       Gluco amylase- beer making and other EtOH products.

       Glucose oxidase- anti oxidant in food processing.

       Invertase- sucrose inversion.

       Lipase- cheese making and flavoring.

       Papain- meat tenderizer and juice clarifier.

       Pectinase- juice clarifier.

       Protease- detergent preparation.

       Rennet- cheese making.

          How proteins are engineered:

        Thermo stable T4 Lysosome: - Changing few amino acids to cysteine that produce more disulfide bonds make the proteins resistant to higher temperatures.  Addition of cysteine does not change the 3-D structure and the function of the protein, yet it is stable at high Tm.

        Thermo stable Triose phosphate isomerase: Increase in temperature leads to deamidation of Aspargine and glutamine.  This deforms the protein and so it looses its activity at higher Tm.  By in vitro site directed mutagenesis amino acid Aspargine at 14 and 78 are changed to Threonine or Isoleucine.  These changes have made the proteins to be thermo stable.

        Cloned b-interferon: Interferons produced in bacteria were not active because of dimerization and

multimerization.  The proteins were found to be inactive.  By in vitro mutagenesis one or two cysteine were changed to serine.  This prevents the individual subunits from dimerization or multimerization, yet they are found to be active.

        Active site modification: Tyrosyl tRNA synthase is the enzyme responsible for adding tyrosine to the tRNA.  Changing the Threonine at 51-st position to Proline has made the enzyme to have increased specific activity and increased specificity.

        Changed Staphylococcus nuclease (S1 nuclease); The S1 nuclease recognizes both ss RNA and ssDNA and dsDNA and acts at A.U or A.T rich regions and cuts to generate 3’phosphate and 5’OH groups.  When Lysine at 116 positions was changed to cysteine, the protein showed remarkable site-specific activity.

        Subtisilin:  It is a bacterial protein used in detergents.  This protein is responsible for removing proteinaceous dirt.  But the protein is susceptible for bleach and it is rendered inactive.  By changing and addition of one or two cysteine residues, the number of S-S bonds increase.  This had made the proteins to be stable not only in the detergent but also stable for bleach.  Such engineered protein is used in detergent powder called biodetergent.

In Vitro Random Mutation:First method:

Using chemicals creates mutation, but the site of mutation is random.  Whether or not in vitro mutation has any desirable feature is perhaps a chance.

In order to induce mutations, obtain ssDNA from M13/18 plasmids with an insert gene of interest.

The ssDNA is subjected very low level of sodium bisulfate under controlled conditions such that the number of site mutated will be very very minimal.  Sodium bisulfate deaminates cytosine residues to uracil.  When the DNA containing uracil replicates it produces DNA with Adenine in place of uracil.  Thus whatever limited numbers of cytosine residues converted into adenines change the message, so also the protein product.

Such deaminated and oxygenated ssDNA is copied into dsDNA by using an universal primer.  Such replicative form DNA is used for bacterial transformation.  Then the colonies are screened for any mutation.  This way one can generate a large number of point mutation.

Second method:

Some of the E.coli strains such as Ung ( – )and Dut(+)  are very useful obtaining in vitro mutation, again it is random.  Ung –

strains are incapable of removing uracil nucleotides from the DNA by deglycosylation reaction.  Dut – strains are lacking UTPase, thus the concentration of UTP inc5reases and UTPs are incorporated into the DNA.  But Ung+ and Dut + strains have functional enzymes.

If a M13/18 plasmid containing a specific gene is used to transform double mutant strain, UTPs are incorporated into the replicating DNA and incorporated UTPs are not removed by N’deglycosylases, because of defective enzymes.

Obtain positive ssDNA from M13/18 transformed strains and using universal primers generate ds DNA.

Digest the plasmid in order to release the insert and clone the same into another plasmid and transform Ung + and Dut – bacterial strains.  As the plasmid DNA replicates all the Us are removed by deglycosylases and the replicated plasmid is obtained.

Obtain the plasmids and release the insert and recline the gene and analyze for the random mutants.

Site directed Mutagenesis: If the sequence of the desired DNA is known, from that one can find out certain restriction enzyme sites.  For example within the gene of interest, assume there is one E.coR I site; the sequence of it is GAATTC.

If the site is cut with EcoR I enzyme it generates 5’ overhangs in both strands of the DNA.

          ----5’G            AATTC---3’

          ----3’CTTAA            G---5’

If such DNA with sticky ends is treated with S1 nuclease it removes the overhangs to blunt ends.

          -----5’G      C---3’

          -----3’C      G----5’

When such ends are ligated one gets the DNA with 5’GC3’ sequences and four base pairs are lost.  This leads not only to deletions but also changes the reading frame.

Similarly if the cut ends are treated with T4 DNA polymerase with dNTPs, the enzyme fills the gaps of each ends by extending the cut ends by 5’3’ direction to produce blunt ends.  If such blunt ends are ligated they generate 5’GAATTAATTC3’.  In this four base pars are added.  Again this causes the addition and the reading frame is changed.  Mostly this kind of changes doesn’t produce any functional protein, if by chance the changed reading frame works you are in luck.

Method II:

In this case the change is directed to one or more specific nucleotides thus one can change only one of the codons.  For example one wants to change Phenyl alanine (UUU) to Cysteine (UGU), design primers in such a way UUU is changed to UGU, where with exception of one nucleotide others are complementary to the nucleotides on either side of the said nucleotide.

          5’--UUU

          3’—AAA

          Primer to the bottom strand- wit5h changed nucleotide-----5’UGU

          Primer to the top strand   with changed nucleotide   ----- -<-ACA 5’

Melt the DNA to ssDNA and anneal the primer to the strands not to a state of stringency.  This will provide an opportunity for the base pairs, which are not complementary, remain unpaired.  Amplify the DNA using PCR protocols.  Then melt the DNA to single strands and anneal them at high Tm for stringency.

Then use the plasmids and transform them and look for the change in the codon.

Another way is to have marker in the DNA which one wants to use for site directed mutation.   Take a plasmid with the desired gene and a marker out side the gene such as a restriction site which can be cut with only Dpn-I.  Use the primers and amplify the DNA.  Then melt the DNA and Anneal.  The parental strands anneal and new strands anneals by themselves.  After annealing the DNA, cut the dsDNA with Dpn-I.  The new strands have no Dpn-I site for the enzyme requires methylated sites, otherwise it does not recognize the site.  Thus the parental strands are cut, but not the new strands.  When such DNA is used for bacterial transformation the cut DNA cannot transform and the uncut

DNA transforms bacteria, thus one obtains the DNA with changed sequence to generate a new codon

Primer extension

BIOC/MCB 568 -- Fall 2010John W. Little--University of Arizona

BIOC/MCB568 Home Page

 Methods

 

Primer extension is used to map the 5' ends of DNA or RNA fragments. It is done by annealing a specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA templates.

 

Uses:

1. Mapping the 5' end of transcripts. This allows one to determine the startpoint of transcription (assuming the mRNA isn't further processed), which helps localize promoters or TATA boxes.

2. Quantifying the amount of transcript in an in vitro transcription system. An example is the Yudkovsky et al. paper.

3. Determine the locations of breaks or modified bases in a mixed population of RNA or DNA samples. This is useful in applications like footprinting. Two different methods are used. In one, the modified nucleotide cannot be recognized by the polymerase or reverse transcriptase; in such cases, the chain ends at the site of modification (as with KMnO4 -- for instance in the Kainz and Roberts paper). In the other, the modification is converted in a later step of the analysis to a strand break by chemical treatment. For instance, the sites of modifications by dimethyl sulfate (DMS) can be identified by treating DNA with DMS, exposing the sample to conditions that break the backbone at the site of modification, followed by primer extension.