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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