MUTAGENESIS AND PROTEIN ENGINEERING

PRESENTED BY

UDO KOKO 146760

EGUN CHRISTA 146035

NNADI SANDRA 147807

OYINLOYE BABATUNJI 147204

ADEYEMO OLUWATOBI 146032

OUTLINEINTRODUCTIONDEFINITION OF MUTAGENESISTYPES OF MUTAGENESISPROCESS/METHODS OF

MUTAGENESISPROTEIN ENGINEERINGCONCLUSION

INTRODUCTION The sequence of a gene dictates the amino acid

sequence of the protein that it encodes.

Mutations within a gene, i.e. alterations to the DNA sequence, may result in changes to the amino acid sequence of the protein.

The analysis of mutations is especially useful in the elucidation of protein function.

Mutations that either reduce the activity of the protein, or allow the protein to behave in an abnormal way, can be used to ascribe particular functions to individual portions of proteins.

INTRODUCTION CONTD. The rate of naturally occurring mutations,

resulting as a consequence of erroneous DNA replication within genes, is quite low (estimated at a level of one DNA base alteration per 106–108 bases replicated)

Naturally occurring mutations have, however, been used to isolate genes and describe specific functions for their encoded proteins. Prior to the explosion in molecular biology techniques in the 1970s and 1980s, increased mutation rates were usually obtained by treating whole cells with either a physical or a chemical mutagen.

CATEGORIES OF MUTATIONMutations within DNA generally fall into one

of two categories; 1) A base or bases within a DNA

sequence are changed from one sequence to another

2) Bases are either inserted into or removed from the gene.

Single DNA base pair changes are

described as being either transition mutations or transversion mutations:

CATEGORIES OF MUTATION CONTD.

• transition mutations – the change of one purine–pyrimidine base pair to a different purine–pyrimidine base pair (e.g. AT→GC, or GC→AT, or TA→ CG).

• transversion mutations – the change of a purine–pyrimidine base pair to a pyrimidine–purine base pair (e.g. AT → TA, or GC → CG, or AT → CG, or GC → TA).

CONSEQUENCES OF MUTATION Single base changes may result in various alterations

such as;1)silent mutation – the triplet code is changed, but the amino acid encoded is the same (e.g. the triplets 5-TCG-3 and 5-TCC-3 both encode the amino acid serine).

2)mis-sense mutation – a codon change alters the amino acid encoded (e.g. if the serine codon 5-TCG-3 is mutated to 5-ACG-3, then the amino acid threonine will be inserted into the encoded polypeptide in place of serine.

3)non-sense mutation – an amino acid codon is changed to produce a stop codon. For example, if the serine codon 5-TCG-3 is mutated to 5-TAG-3, then the encoded polypeptide chain will terminate at this point.

CONSEQUENCES OF MUTATION CONTD.

The insertion or deletion of a base pair, or base pairs, into the coding sequence of a gene can have drastic implications for the encoded polypeptide. Since the DNA code is read in triplets, the insertion or deletion of bases in multiples other than three will result in a frame-shift mutation.

MUTAGENESIS Mutagenesis is an induced form of

mutation It could be invivo or invitro Invivo mutagenesis occurs when an

organism is exposed to physical or chemical e.g x-rays, UV light or chemical such as ethyl methane sulphonate (EMS) to generate DNA bases throughout the genome.

Invitro mutagenesis involve changes that may be localized or general, random or targeted.

TYPES OF MUTAGENESISSite Directed Mutagenesis

Saturation Mutagenesis

PROCESS/METHODS OF MUTAGENESIS

Primer Extension Mutagenesis (Site Directed Mutagenesis)

Cassette MutagenesisPCR based MutagenesisQuick change MutagenesisAlanine Scanning Mutagenesis

Primer Extension Mutagenesis The use of oligonucleotides in creating site-

directed mutations was devised in the laboratory of Michael Smith, who shared the 1993 Nobel Prize in Chemistry for his discovery.

Principle/procedure:The oligonucleotide binds to its complementary

sequence within the single stranded genome, and is designed such that one or more mutations(non-complementary base pairings) occur when it binds to the M13 DNA. The binding of the oligonucleotide to the single-stranded DNA is stabilized by the complementary base pairing that occurs elsewhere.

PRIMER EXTENTION CONTDOnce bound to its complementary

sequence, the oligonucleotide provides a free 3 hydroxyl group as the starting point of DNA synthesis.

The newly synthesized DNA circle is then completed by the action of DNA ligase, in the presence of ATP, to seal any nicks remaining in the DNA backbone.

The naked DNA is unable to infect E. coli cells, so in it must be introduced to the bacterium where the DNA will be replicated and phage particles produced.

Fig 1.Site directed mutagenesis using a single-stranded DNA template

CASSETTE MUTAGENESISCassette mutagenesis relies on the presence of two

restriction enzyme recognition sites flanking the DNA that is to be mutated.

The plasmid is cut with the enzymes,EcoR1 and Pst1.

The linear plasmid DNA is then ligated to a synthetic double-stranded DNA produced through the annealing

of two complementary oligonucleotides. The complementary oligonucleotides contain the

desired mutation(s) and the required overhanging sequences for the ligation to the restriction enzyme cleavage sites (Wells,Vasser and Powers, 1985).

Fig. 2.0 Cassette mutagenesis

PCR BASED MUTAGENESISBy suitable design of oligonucleotide

primers, mutations can be introduced into the

ends of PCR products in a way that leads to mutagenesis efficiency of almost 100 per cent.

But mutation limited to the 5’ ends of the strands, to enable the creation of mutation at any point through the length of the PCR product, method called a two-step PCR mutagenesis was developed.

PCR BASED MUTATION CONTD. Requires four oligonucleotide primers and three separate

PCR reactions and is outlined in Figure 3.0.

Two of the primers (1 and 4) are designed to be complementary to the anti-sense strand and the sense strand of the target DNA, respectively.

The other two primers (2 and 3) are designed to bind to the different strands of the same DNA sequence and will also introduce the required mutation into each strand.

In the first PCR, the 5-end of the gene is amplified using primers 1 and 2. The resulting product will bear the mutation at its 3-end.

PCR BASED MUTAGENESIS CONTD.

In the second PCR, the 3-end of the gene is amplified using primers 3 and 4 so that the resulting product will bear the mutation at its 5-end.

Primers 2 and 3 are designed such that they are complementary to each other and overlap with one another. This means that the 3-end of PCR product 1 will be identical to the 5-end of PCR product 2.

Therefore, if PCR products 1 and 2 are mixed with

each other, denatured and allowed to cool, then the individual strands from each reaction can hybridize with each other.

Two possible hybrid molecules can form.

Fig 3.0. A Two-step PCR mutagenesis protocol(Higuchi et al,1988)

QUIKCHANGE MUTAGENESISThis is a method using the power of PCR

to introduce mutations directly into plasmid DNA

This method utilizes two oligonucleotide primers

One of the primer is produced so it is complementary to the sense strand of the gene and contains the desired mutation,

While the other primer is designed to be complementary to the anti-sense strand of the gene, but also contain mutation.

QUIKCHANGE MUTAGENESISCONTD.

Double stranded plasmid DNA is used as a PCR template.

The primers are extended in a PCR reaction to synthesize both plasmid DNA strands, each of which contains the mutation.

The DNA is then digested with the restriction enzyme DpnI, which can only cleave methylated DNA (wild type) without affecting the non methylated DNA (mutant).

QUIKCHANGE MUTAGENESIS

Fig. 4.0 Quikchange mutagensis, (Wang and Malcolm, 1999)

QUIKCHANGE MUTAGENESISCONTDThis procedure is very rapid (3–4

hours)

Highly efficient (∼80 per cent) at producing mutant DNA plasmids

It does require additional cloning steps.

PROTEIN ENGINEERING

Protein engineering can be thought of as the deliberate modification of the sequence of a protein (through the alteration of the DNA sequence encoding it) to impart the protein with a new or novel function.

This approach has been used for the creation of enzymes with altered characteristics that may be desirable for particular purposes.

The sorts of enzyme characteristics that may be altered include.

PROTEIN ENGINEERING CONTD.

Thermal stability pH stability Kinetic properties Stability in organic solvents Altered cofactor requirement Altered substrate binding

specificity Resistance to proteases Changed allosteric regulation.

CONCLUSIONThe ability to introduce mutations at

will within a segment of DNA has allowed many exceptionally elegant and precise gene analyses to be performed that would have not been previously possible.

Sequel to this, we have the opportunity to design and mass produce novel proteins that are different from those made by the living organisms.

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