site-directed mutagenesis

Examples of site-directed mutagenesis in the following topics:

• Mutation

  • Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.
  • In general, this form of mutagenesis requires that the wild type gene sequence be known.
  • The copied gene contains the mutated site.
  • Many approaches have since been developed to improve the efficiency of mutagenesis.
  • In this experiment, random mutations were introduced into the virus by site-directed mutagenesis.

• Shuttle Vectors and Expression Vectors

  • Expression vectors are used for molecular biology techniques such as site-directed mutagenesis.
  • They can also be used for in vitro experiments and modifications such as mutagenesis and PCR.
  • Please note the presence of a multiple cloning site, a promoter, a repressor, and a selectable marker.

• Coupling Specific Genes to Specific Organisms Using PCR

  • Through site-directed mutagenesis or customized primers, individual mutations in DNA can be made.

• Inactivating and Marking Target Genes with Transposons

  • Insertional mutagenesis is a technique used to study the function of genes.
  • Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome.
  • An alternative strategy for insertional mutagenesis has been used in vertebrate animals to find genes that cause cancer.
  • Specifically, the transposon contains signals to truncate expression of an interrupted gene at the site of the insertion and then restart expression of a second truncated gene.

• Additional Resources

  • Critical Issue: Working Toward Student Self-Direction and Personal Efficacy as Educational Goals: Collection of many resources (including video clips) on how to enhance student self-efficacy (http://www.ncrel.org/sdrs/areas/issues/students/learning/lr200.htm).
  • Information on self-efficacy: Professor Albert Bandura's web site on self-efficacy.
  • This site collects many learning theories and models in relation to self-efficacy http://www.des.emory.edu/mfp/self-efficacy.html#bandura).

• Know what your customers want

  • Recognize the needs of all who will use your site, vendors, customers and yes even employees, and direct each to the content that will interest them most.
  • One such site, video how-to site http://www.Howcast.com, is a gem.
  • They can visit your site and be taken to useful, informative sites.
  • Note: a number of these sites are fee based, the more times your site comes up in a search the more it may cost you.
  • Both of these sites as well as MSN.com are the most commonly used web search engines sites globally.

• The Mechanism of Protein Synthesis

  • Once at the cap, the pre-initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon.
  • The intact ribosome has three compartments: the A site binds incoming aminoacyl tRNAs; the P site binds tRNAs carrying the growing polypeptide chain; the E site releases dissociated tRNAs so that they can be recharged with amino acids.
  • The aminoacyl-tRNA with an anticodon complementary to the A site codon lands in the A site.
  • The E site moves over the former P-site tRNA, now empty or uncharged, the P site moves over the former A-site tRNA, now carrying the growing polypeptide chain, and the A site moves over a new codon.
  • After the peptide bond is created, the growing polypeptide chain is attached to the A site tRNA, and the tRNA in the P site is empty.

• Gene Inversion

  • Recombining sequences in site-specific reactions are usually short and occur at a single target site within the recombining sequence.
  • For this to occur, there is typically one or more cofactors (to name a few: DNA-binding proteins and the presence or absence of DNA binding sites) and a site specific recombinase.
  • Fimbrial adhesion by the type I fimbriae in E. coli undergoes site specific inversion to regulate the expression of fimA, the major subunit of the pili, depending on the stage of infection.
  • The FimE recombinase has the capability to only invert the element and turn expression from on to off, while FimB can mediate the inversion in both directions.

• Electrophilic Substitution of Disubstituted Benzene Rings

  • The site at which a new substituent is introduced depends on the orientation of the existing groups and their individual directing effects.
  • Symmetry, as in the first two cases, makes it easy to predict the site at which substitution is likely to occur.
  • Note that if two different sites are favored, substitution will usually occur at the one that is least hindered by ortho groups.
  • In examples 4 through 6, oppositely directing groups have an ortho or para-relationship.
  • Example 6 is interesting in that it demonstrates the conversion of an activating ortho/para-directing group into a deactivating meta-directing "onium" cation [–NH(CH3)2(+)] in a strong acid environment.

• Regulatory Mechanisms for Cellular Respiration

  • Some reactions are controlled by having two different enzymes: one each for the two directions of a reversible reaction.
  • In contrast, if two different enzymes (each specific for a given direction) are necessary for a reversible reaction, the opportunity to control the rate of the reaction increases and equilibrium is not reached.
  • A number of enzymes involved in each of the pathways (in particular, the enzyme catalyzing the first committed reaction of the pathway) are controlled by attachment of a molecule to an allosteric (non-active) site on the protein.
  • This site has an effect on the enzyme's activity, often by changing the conformation of the protein.
  • The attachment of a molecule to the allosteric site serves to send a signal to the enzyme, providing feedback.