Mutagenesis
What Is a Mutation? Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive.
Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations in specific ways and then observing the phenotype of the organism the function of genes and even individual nucleotides can be determined.
Some types of Mutagenesis Directed Mutagenesis Site-directed/Site-specific Mutagenesis Mismatched Mutagenesis
Directed Mutagenesis a largely discredited hypothesis proposing that organisms can respond to environmental stresses through directing mutations to certain genes or areas of the genome.
Site-directed Mutagenesis Where a specific site in a cloned DNA needs to be altered in a precise, predetermined way Can be designed to create specific nucleotide substitutions, deletions, and so on
Image of Site-directed Mutagenesis
Site-directed mutagenesis Juang RH (2004) BCbasics Wild type CAG GTC CAG GTC (1) (5) translation (2) CAG + primer Val Wild type protein Mutant primer (3) CAG GCC + polymerase (6) CGG GCC translation CAG GCC (4) replication Mutant protein Thr Only one amino acid changed Smith (1993) Val Thr
Two different forms of Site-specific Mutagenesis 5 add-on mutagenesis - Diagram on page 148 of the text - a new sequence or chemical group is added to the 5 end of a PCR product Mismatched primer mutagenesis - primer is designed to be only partially complementary
How Do We Fix Mutations? Site-Directed Mutagenesis! Start a project!! -Start by creating oligonucleotides which will introduce the original base to the mutated DNA and replace wrong the base.
Oligonucleotides are Synthetic single-stranded fragments of DNA used for priming the mutated clones. In order to work, the primers must meet the following criteria: -must contain desired mutation -mutation should be in the middle of the primer -the GC content should be at a minimum of 40% and should terminate in one or more of C or G bases Now, onto the process of mutagenesis
Thermal Cycling -The mutagenic primer anneals to the parental DNA plasmid. -The DNA polymerase extends and incorporates the mutagenic primers resulting in nicked circular strands.
Results of Thermal Cycling with This is a Gel picture showing the results of Thermal Cycling with the KOD polymerase. The arrow is pointing to the clone we are tracking throughout this process. (364G11.x2) KOD
Results of Thermal Cycling with After the final analysis of the clones made with KOD, there was a problem. The mutation was fixed, but the sequence of some clones had a tandem repeat. So we decided to use Pfu Turbo polymerase. This is the Gel picture. Pfu Turbo
Dpn1 is an enzyme that digests the methylated, or the parental template, DNA. It leaves the DNA that contains the desired mutation, which is in the correct sequence. Dpn1 Digestion
Transformation, Extraction, and Sequencing We transform the un-methylated DNA using competent cells. The bacterial colonies are picked then prepped to extract the DNA from the bacteria. The DNA is sequenced using the sequencing machine. The tangible product is the computer analysis
Results of Sequencing After all the processes, we have a fixed clone. Mutagenesis was successful.
Mutated Sequence vs. Fixed Sequence
Mismatched Mutagenesis Diagram pg. 147 of text; Figure 5.19 Can create a desired point mutation at a unique predetermined site within a cloned DNA molecule At the intended mutation site it bears a base that is complementary
Benefits of Mutagenesis Well, besides the fact that it fixes mutations in our clones, why even resort to mutagenesis? -Each month, a certain quota must be met, the process of mutagenesis ensures productivity. Since it is a process all it s own, all the work of mutagenesis can be concentrated on by one person. Time efficient. Mutagenesis is a lot simpler than the usual process of cloning.