Recombinant DNA and Biotechnology

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1 Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study DNA Function? What Is Biotechnology? How Is Biotechnology Changing Medicine, Agriculture, and the Environment? Recombinant DNA DNA molecule made in the laboratory that is derived from at least two genetic sources Restriction enzymes (endonucleases) DNA ligase splice DNA fragments together in new combinations (never existed before) Cutting and splicing DNA How do we insert new genes? Recombinant DNA technology can be used to clone, or make identical copies of genes Transformation: Recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal) The altered host cell is called transgenic Need selectable markers To insert genes Changes in membrane permeability Electroporation Chemical treatment

2 Use of vectors (virus, bacteria) New DNA must be able to replicate inside the cell: Replicon or replication unit Plasmids are good vectors Small Easy to manipulate One or more restriction sites (occurring only once) Genes for antibiotic resistance (becoming selectable markers) Have an ori (origin of replication) Replicate independently from host chromosome Plasmid as a vector Eukaryotic genes are too large to be inserted into a plasmid Viruses can be used (bacteriophage) T DNA genes are removed and replaced with foreign DNA. Altered Ti plasmids transform Agrobacterium cells, then the bacterium cells infect plant cells. Whole plants can be regenerated from transgenic cells, or germ line cells can be infected. Not many cells take up the vector (1 cell in 10,000) Selectable marker is antibiotic resistance Easy to use two different antibiotics Selectable markers are called reporter genes Identifying the cells with the vector Remember your glowing colonies? Sources of dna for cloning Gene libraries Reverse transcription from mrna

3 Products of PCR Artificial synthesis or mutation of DNA Genomic library Collection of DNA fragments representing the genome of an organism If phage λ is used as a vector, about 160,000 volumes are required to store the library. One petri plate can hold thousands of phage colonies, or plaques. DNA in the plaques is screened using specific probes Complimentary DNA (CDNA) mrna is extracted from cells, then cdna is produced by complementary base pairing, catalyzed by reverse transcriptase. A cdna library is a snapshot of the transcription pattern of the cell. cdna libraries are used to compare gene expression in different tissues at different stages of development Pcr & synthetic dna DNA can be amplified by PCR Synthetic DNA (can be manipulated to create specific mutations) Manipulating dna Knock out experiments Complementary RNA DNA microarrays Knock out mouse Complementary rna Translation of mrna can be blocked by complementary micro RNAs antisense RNA.

4 Antisense RNA can be synthesized and added to cells to prevent translation the effects of the missing protein can then be determined. Interference rna irna natural mechanism that blocks translation Can be made synthetically in lab (small interfering RNA, sirna) Antisense RNA and RNAi are also used to study cause-and-effect relationships. Example: Antisense RNA is used to block translation of proteins essential for growth of cancer cells. Antisense rna Dna microarray Provides a large array of sequences for hybridization experiments. DNA sequences attached to glass wells Can be used to identify specific single nucleotide polymorphisms or other mutations. If mrna is to be analyzed, it is usually incubated with reverse transcriptase to make cdna. Fluorescent dyes tag the cdnas and they are used to probe the DNA on the microarray. Dna on a chip Microarray to study breast cancer prognosis biotechnology Use of living cells or organisms to produce materials useful to people. Many uses: wine making, yogurt, alcohol, antibiotics, etc Expression vectors needed to express the desire gene in the host For eukaryotes: The poly A-addition sequence, transcription factor binding sites, and enhancers, must be included Expression of a transgene in host Uses of biotechnology Tpa use for stroke victims pharming Production of pharmaceuticals in farm animals or plants

5 Example: Transgenes are inserted next to the promoter for lactoglobulin a protein in milk. The transgenic animal then produces large quantities of the protein in its milk. Growth hormone production (in transgenic cows) pharming Genetically modified foods Agricultural uses Agricultural uses Transgenic cotton, soybeans, tomatoes, corn are being grown so less pesticide is needed Some are being made herbicide resistant Rice does not have β-carotene, but does have a precursor molecule Genes for enzymes that synthesize β-carotene from the precursor are taken from daffodils and inserted into rice by the Ti plasmid Transgenic rice Biotechnology use for environment Making plants slat-tolerant Instead of manipulating the soil or environment we can manipulate the plants Improving environmental conditions: BIOREMEDIATION (clean up oil spills with oileating bacteria) Salt-tolerant tomato plants Concerns with biotechnology Genetic manipulation is an unnatural interference in nature Genetically altered foods are unsafe to eat Genetically altered crop plants are dangerous to the environment Concern over environmental effects centers on escape of transgenes into wild populations: For example, if the gene for herbicide resistance made its way into the weed plants. Advocates for biotechnology All crop plants have been manipulated by humans Since only single genes for plant function are inserted into crop plants, they are still safe for human consumption Genes that affect human nutrition may raise more concerns

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