Biotechnology is the use of microbes to make practical products. The top three justifications for manipulating the genomes of cells are:
1. to eliminate undesirable phenotypic traits in humans, animals, plants and microbes.
2. to combine the beneficial traits of two or more organisms to create a new more valuable organism.
3. to create cells that synthesize products humans need.
Recombinant DNA technology employs a number to tools and techniques to isolate genes and insert them into cells grown in culture. The following are five tools of recombinant DNA technology and examples of application of the tools.
Mutagens are physical and chemical agents that produce mutation. Some mutations are beneficial, so creating a large number of mutations increases the probability of discovering a beneficial mutation. For example the fungus Penicillium may be mutated to syntheisize a more effective antibiotic. Mutagens include uv radiation and ionizing radiation. Mueller first demonstrated the effects of X rays on Drosophilia, establishing cause and effect between radiation and genetic changes manifested in phenotypic changes. Chemical mutagens in bacteria may be carcinogenic in humans. The Ames test utilizes bacteria to test for mutagenic activity, and identify chemicals that may induce cell changes that lead to cancer. Ethidium bromide which may be used to visualize DNA on gel, is also a mutagen because it disrupts base pairing.
1. Explain and describe one physical and one chemical mutagen and its application.
Reverse transcriptase synthesizes cDNA from mRNA, which is the reverse of information flow as depicted in the central dogma of molecular biology, i.e. DNA is transcribed to RNA which is translated to peptides. Cells may have millions of copies of mRNA, so being able to synthesize the complementary DNA reveals the gene that is being expressed. In addition, cDNA contains no introns because of processing in the eukaryotic transcription, so that cDNA may be inserted into a prokaryotic cell and may be translated into the corresponding peptide.
2. Explain how reverse transcriptase differs from RNA polymerase. Give one example of an application of reverse transcriptase in recombinant DNA technology.
Synthetic nucleic acids are synthesized in vitro using enzymes from cells. Currently there are machines that syntheized DNA or RNA by having the sequence of bases entered tthrough a keyborad.
3. If each letter of the keyboard represents a base in DNA, how many keys does the keyboard require? How does this means of DNA synthesis differ from DNA replication in the cell?
Synthetic nucleic acids were used to elucidate the genetic code, create genes for specific proteins, synthesize probes to locate genes in a genome, to synthesize antisense nucleic acids and to make PCR primers.
4. What is the genetic code, how might a synthetic nucleotide be used to determine which amino acid corrresponds to which codon?
5. Give an example of a probe with a flourescent tag that will be used to locate a gene associated with an aggressive form of breast cancer.
Restiction enzymes cut dsDNA at restriction sites, or palindromes. Examples of restriction enzymes are EcoRI and HindIII. There are hundreds of restriction enzymes isolated from bacteria and used to cut DNA at predictable sites. One type of restriction enzyme cuts dsDNA to make blunt ends and the other type creates sticky ends.
6. Compare and contrast blunt and sticky ends, and give one example of each type of restriction enzyme.
Vectors insert DNA into a new cell so that the cell acquires a new phenotype. Vectors are pieces of DNA that are small enough to manipulate in a lab, survive inside the new cell, contain a recognizalbe genetic marker and carry the gene regions necessary to ensure the gene is transcribed and translated. Examples of vectors are plasmids, viruses and transposons.
7. Explain one risk of a viral vector that inserts itself into a necessary gene and causes a mutation.
Gene library is a collection of cells or viruses, in which each member carries a portion of a given organism’s genome. Gene libraries provide a ready
Techniques of recombinant DNA technology include the polymerase chaing reaction (PCR) developed by Cary Mullis. PCR has three steps that are repeated while the same sample of primers, target gene, taq polymerase and nucleotides are cycled through three different temperatures in a microfuge tube. Gel electrophoresis separated fragments of molecules by size, shape and charge. DNA microarrays are able to monitor thousands of genes on one plate. Applications of these techniques include diagnostics or vaccine design.
8. Explain how a subunit vaccine is designed using genes from a pathogen and a viral vector? p502-504.
One proposed way to stop the spread of arboviruses by mosquitoes is to vaccinate the mosquito. This type of vaccine is a transmission blocking vaccine (TBV). One typeof TBV vaccinates the human so that a blood meal from a vaccinated human would prevent the mosquito from being infected. Another strategy is to use the gene drive known as Crispr-Cas9 which would edit the mosquito genome to confer resistance to diseases mosquitoes transmit to humans, and is very targeted. This requires wild type mosquitoes to mate with lab mosquitoes that have been genetically modified. Some data would indicate the lab mosquitoes are not competitve with wild type males.
9. Explain one advantage and one disadvantage of the TBV approach for controlling the spread of arboviruses.
10. Does the public have any control over the use of gene drives to alter species in the ecosystem for public health concerns?