SEMINAR PRESENTATION

TOPIC: MUTAGENESIS TECHNIQUES

PRESENTER: WEREMA CHACHA

DATE: FRIDAY, 24TH DECEMBER, 2010

VENUE: L 406

TIME: 9:20 – 9:40 AM

Introduction

• Mutagenesis is a process by which the genetic

information of an organism is changed in a

stable manner, either in nature or

experimentally by the use of chemicals or

radiations.

• Mutagenesis as a science was developed

especially by Charlotte Auerbach in the first

half of the 20th century.

• There are two major classes of mutation i.e.

gene and chromosomal mutations.

Importance of mutagenesis

• Beneficial mutations i.e. evolution

• Harmful mutations i.e. cancer – due to damage

of genes controlling the cell cycle.

Types of mutagenesis techniques

• There are the following types of mutagenesis:

• Site – directed mutagenesis

• Transposon mutagenesis

• Signature tagged mutagenesis

• This presentation will focus on site-directed

mutagenesis

Site-directed mutagenesis

• Site-directed mutagenesis, also known as

site-specific mutagenesis or oligonucleotide-

directed mutagenesis, is a molecular biology

technique in which a mutation is created at a

defined site in a DNA molecule.

• Recently, site-directed mutagenesis has

become one of the most commonly used

methods in molecular biology.

Principles

• In general, this form of mutagenesis requires

that the wild - type gene sequence be known.

• Thus, enables the mutant oligonucleotides or

primers to be synthesized.

TYPES

• There are three common different methods of

site-directed mutagenesis namely;

• Cassette mutagenesis

• Primer extension and

• Procedures based on the PCR.

Cassette mutagenesis

• In cassette mutagenesis, a synthetic DNA fragment containing the desired mutant sequence is used to replace the corresponding sequence in the wild-type gene.

• It is a simple method for which the efficiency of mutagenesis is close to 100%.

• The disadvantages are the requirement for unique restriction sites flanking the region of interest.

Cont..

• The limitation on the realistic number of

different oligonucleotide replacements that can

be synthesized.

• The latter problem can be minimized by the

use of doped oligonucleotides.

• Doped oligonucleotides usually refers to the

use of unequal amounts of each of the four

standard dNTPs in oligonucleotide synthesis.

Primer extension:

single-primer method

• The simplest method of site-directed mutagenesis is the single-primer method (Gillam et al. 1980, Zoller & Smith 1983).

• The method involves priming in vitro DNA synthesis with a chemically synthesized oligonucleotide (7–20 nucleotides long) that carries a base mismatch with the complementary sequence.

• The method requires that the DNA to be mutated is available in single-stranded form, and cloning the gene in M13-based vectors makes this easy.

Cont..

• However, DNA cloned in a plasmid and obtained

in duplex form can also be converted to a partially

single-stranded molecule that is suitable.

• The synthetic oligonucleotide primes DNA

synthesis and is itself incorporated into the

resulting heteroduplex molecule.

• After transformation of the host E. coli, this

heteroduplex gives rise to homoduplexes whose

sequences are either that of the original wild-type

DNA or that containing the mutated base.

Cont..

• The frequency with which mutated clones arise, compared with wild-type clones, may be low.

• The major reason for this low yield of mutant progeny is that the methyl directed mismatch repair system of E. coli favors the repair of non-methylated DNA

• In the cell, newly synthesized DNA strands that have not yet been methylated are preferentially repaired at the position of the mismatch, thereby eliminating a mutation.

Cont..

• In a similar way, the non-methylated in vitro-

generated mutant strand is repaired by the cell

so that the majority of progeny are wild type

(Kramer, B. et al. 1984).

• The problems associated with the mismatch

repair system can be overcome by using host

strains carrying the mutL, mutS or mutH

mutations, which prevent the methyl-directed

repair of mismatches.

PCR methods of site-directed

mutagenesis

• PCR is a primer-mediated enzymatic amplification of specifically cloned or genomic DNA sequences.

• It has become a routine procedure in every molecular biology lab for manipulating and identifying genetic material.

• This technique can be used to identify with a very high-probability, disease-causing viruses ,protozoa and/or bacteria, a deceased person, or a criminal suspect.

Cont..

• Early work on the development of the PCR method of DNA amplification showed its potential for mutagenesis.

• Single bases mismatched between the amplification primer and the template become incorporated into the template sequence as a result of amplification.

• TTAACGGGGCCCTTTAAA........TTTAAACCCGGGTTTAATTACCCCGGGAAATTT.......................>

<..............................................TTTAAGCCCGGGTTTAATTGCCCCGGGAAATTT........AAATTTGGGCCCAAA

Diagram - PCR Cycle Steps

Cont..

• Higuchi et al.(1988) have described a variation of the basic method which enables a mutation in a PCR-produced DNA fragment to be introduced anywhere along its length.

• Two primary PCR reactions produce two overlapping DNA fragments, both bearing the same mutation in the overlap region.

• The overlap in sequence allows the fragments to hybridize.

Cont..

• This method can generate mutations (base

substitutions, insertions, and deletions) from

double-stranded plasmid without the need for

subcloning into M13-based bacteriophage

vectors and for ssDNA rescue.

• The advantage of a PCR-based mutagenic

protocol is that the desired mutation is obtained

with 100% efficiency.

Cont..

• Disadvantages; PCR product usually needs to be ligated into a vector, although Sarkar and Sommer (1990) have generated the mutant protein directly, using coupled in vitro transcription and translation.

• Taq polymerase copies DNA with low fidelity.

• Therefore the sequence of the entire amplified segment generated by PCR mutagenesis must be determined to ensure that there are no extraneous mutations.

• Alternatively, thermostable polymerases with improved fidelity can be used.

Mutagenesis applications

• In-vitro site-directed mutagenesis researches

leads to the following;

• Used to study protein structure and functions.

• Identify enzymes active sites.

• Design novel protein in vaccine and drug

discovery.

References

• Primrose S.B., Twyman M. R., and Old W.R. (2001). Principle of gene

manipulation sixth edition; Chap. 7, p. 132-138

• Higuchi, R., Krummel, B. and Saiki, R. (1988) Nucl. Acids Res. 16, 7351.

• Sarkar, G. and Sommer, S.S. (1990) BioTechniques 8, 404.

• Neal Cosby and Scott Lesley (1997). Promega Notes Magazine Number

61, p. 12

• Jenkins G.J.S., Suzen H.S., Sueiro R.A.,and Parry J.M (199). The

restriction sites mutations assay; Mutagenesis vol. 14 no.5 p. 439 – 448

• Timothy D. P. and Robin S. K. (1994) Transposon Mutagenesis of

Rhodobacter sphaeroides, Chap. 3, p.46-58

• Holden, D. W. and Hensel M. (1998).Signature tagged mutagenesis.

Methods in Microbiology 27:359-370.

• Petra S.,M onica L., Eric F. J., and Byron K. (1993). Cassette Mutagenesis

of a Potential Substrate Recognition Region of Cytochrome P450 2C2. The

journal of biological chemistry VOl. 268, No. 29, pp. 21997-22003

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