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Troubling and Terrific Technology Chapter 20 I. Introductions to DNA Technology A. Biotechnology - manipulation of organisms or their components to make useful products. used to make hundreds of products can insert genes from one species into another to make desired proteins used in solving crimes used in agriculture I. Introductions to DNA Technology B. C. Recombinant DNA - DNA in which genes from two different sources (can be different species) are combined in vitro into the same molecule Genetic Engineering - direct manipulation of genes for practical purposes II. Gene cloning Preparing well-defined, gene sized pieces of DNA in multiple copies. Can make copies of gene (ex. pest resistance gene, sequencing) Can make protein from the gene (ex. make growth hormone) A. General Method for gene cloning 1. 2. 3. 4. 5. 6. Isolate the DNA of a plasmid and the DNA containing the gene you want to clone Insert the gene into the plasmid (recombinant DNA) Put plasmid back into bacteria (recombinant bacteria) Allow bacteria to replicate and clone gene Identify bacteria that have the clone Many applications: make copies of gene, make protein from the gene B. Restriction Enzymes to make Recombinant DNA 1. Characteristics and Terms enzymes that cut DNA molecules at a limited number of specific locations used by bacteria in nature to cut up foreign DNA bacterial DNA is not destroyed because it has methyl groups added where restriction enzymes work B. Restriction Enzymes to make Recombinant DNA Restriction site - recognition sequence for restr. enzyme (the place where it will cut. Restriction fragments - when DNA is exposed to a restriction enzyme, the enzyme cuts it in the same place every time. This leaves the same fragments of DNA every time it is exposed to the restriction enzyme Sticky ends - restriction fragments can be staggered, leaving short stretches of single stranded DNA (sticky ends). These single stranded areas can hydrogen bond to different DNA that has been cut with the same restriction enzyme. (temporary bond) B. Restriction Enzymes to make Recombinant DNA DNA ligase - can be added to mixtures of DNA to make the temporary bonds permanent Cloning vector - DNA molecule (usually a plasmid) that can carry foreign DNA into a cell and replicate (usually in a bacterial host) 2. How do you make a Bacteria express a eukaryotic gene??? Problems in Paradise! Problem 1: gene expression is different in prokaryotes and eukaryotes Solution: Use an expression vector - a cloning vector that contains a promoter region upstream of the restriction site When you insert the eukaryotic gene, the bacteria’s promoter will function and express the foreign gene Problem 2: lots of non-coding regions (introns) in eukaryotic genes (bacteria don’t have spliceosomes) Solution: make artificial eukaryotic genes without introns allow transcription of original gene to premRNA RNA splicing occurs mRNA is released from nucleus isolate mRNA from cell add reverse transcriptase (makes DNA from RNA) the DNA made is called cDNA (complementary) and contains only the gene of interest (no introns) cDNA will synthesize complementary strand Use cDNA to insert into bacteria DNA Libraries Genomic libraries are sets of plasmid containing cell clones Bacterial artificial chromosomes (large plasmids) contain genes for replication and carry larger inserts cDNA libraries contain genes that have already been transcribed To find the correct gene from the library – use nucleic acid hybridization with a Probe that is tagged (usually radioactively) 3. Other ways to avoid prokaryotic/eukaryotic problems Use Eukaryotic cell as host - YEAST is a great eukaryotic host: it grows quickly, is single celled and even contains plasmids To get eukaryotes to take up DNA, scientists can use electroporation - a brief electrical pulse that makes a temporary hole in the cell membrane or they can physically inject DNA using a microscopic needle C. PCR - Polymerase Chain Reaction method to copy any piece of DNA (even small quantities) without using cells incubate DNA in test tubes with DNA polymerase (special type able to withstand high heat), short pieces of ssDNA for primers, and a supply of nucleotides (to make new DNA) useful for making a limited quantity of DNA (for testing crime scenes, diagnosing prenatal diseases, mammoth cloning etc) 6. Gel Electrophoresis separates macromolecules (nucleic acids and proteins) based on size, electrial charge and other physical properties DNA (separated by size) is sorted into a mixture of bands of DNA that are the same length (negatively charged DNA moves toward positive end of gel) Can be used to compare genes in individuals or between species A. Procedure 1. 2. Prepare restriction fragments Mix DNA samples to be tested with the same restriction enzyme Electrophoresis Put the samples into different wells of a gel (see picture) Attach electrodes at both ends. DNA has a negative charge and will be attracted to the positive end of the gel Longer molecules move more slowly through the gel and shorter molecules move quickly Each sample will produce a different banding pattern A. Procedure 3. Southern Blotting 4. Transfer DNA from gel to special nitrocellulose paper This denatures the DNA and makes it single strands Hybridization Expose the paper to radioactively labeled probe that is complementary to the sequence you are trying to find Even if you start with an entire genome (i.e. all 46 chromosomes), only the sequence you are looking for will be hybridized A. Procedure 5. Autoradiography put photographic film over the paper. radioactivity exposes the film and forms an image banding patterns are produced RFLP - restriction fragment length polymorphisms Differences found in non-coding regions of DNA Serves as a genetic marker for a particular location These markers can have many variants within a species (like different phenotypes) These can serve as genetic markers used to make linkage maps (like the fruit fly maps we made before) The more often a RFLP and a gene are inherited together, the closer they are on a chromosome Human Genome Project An effort to map the entire human genome, determining the complete nucleotide sequence of the DNA of each human chromosome. Done using three methods 1. Genetic (linkage) mapping make a linkage map of several thousand genetic markers spread evenly throughout the chromosomes markers can be genes, satellite DNA or RFLP’s markers used as a reference point for mapping 2. Physical mapping ordering DNA fragments distance between markers expressed in a physical measure done by cutting DNA with restriction enzymes and determining the order of the fragments produced must make fragments that overlap and use probes to find overlap 3. DNA sequencing Use the technology of cloning fragments of DNA use a sequencing machine that combines DNA labeling, DNA synthesis and electrophoresis A. Sanger method= prepare a single stranded DNA with unknown sequence, divide it into 4 portions B. Incubate with all necessary components to make new DNA strand 3. DNA sequencing C. Put the mixtures into four different tubes with special nitrogen bases called didoxyribonucleotides (ddATP, ddCTP, ddTTP, ddGTP) D. When a dd____ nucleotide is added, the strand stops growing. This happens at random points along the new DNA strand E. Eventually, a set of labeled strands of various lengths is generated and can be run and separated on a gel F. The bands on the gel are in direct correspondence with the sequence of DNA IV. How is all this info used??? A. Analyzing DNA sequences- scientists can use computers to exam stretches of DNA and find the parts that actually code for genes (look for start/stop codons etc) Belief now is that there are only 30-40000 genes - most of our genome is non coding Most vertebrate genes can code for 2 or 3 polypeptides by changing the splicing of mRNA How is all this info used??? B. Comparing genes - scientists are using DNA to compare genes in different organisms and within the same species Many genes are seen in multiple organisms even those very distantly related C. Studying gene expression - DNA can be used to see which genes are active in which cells, and when in development these genes might be turned on and off D. In vitro mutagenesis - if scientists don’t know the function of a gene they have found, they can mutate the gene and look for changes in the cell or organism Cloning Producing one or more organisms that is genetically identical to the parent Plants – can be cloned by growing from cuttings Used to reproduce plants with favorable characteristics like pest resistance Cloning - Animals Done by nuclear transplantation Remove nucleus of unfertilized egg Replace it with nucleus from cell you are cloning Let it divide in culture Put egg in surrogate mother May lead to cells that are not as healthy as normal cells Can lead to other problems like obesity, pneumonia, liver failure, premature death (all these found in cloned mice) Stem Cells Relatively unspecialized cell that can reproduce itself and can differentiate into specialized cells Embryonic Stem cells – come from early embryo Reproduce indefinitely Can become variety of specialized cells, even eggs and sperm Adult Stem Cells – used to replace nonreproducing specialized cells as needed Practical Applications A. Diagnosis of Diseases - using PCR and probes, certain pathogens can be detected *hundreds of diseases are diagnosed using this technology i.e. HIV, hemophilia, cystic fibrosis B. Human Gene Therapy - alteration of problem genes - may be possible by replacing or repairing the problem gene i.e. defective bone marrow cells repaired and returned to patient. As the cells divide, regular bone marrow cells are formed Practical Applications C. Pharmaceutical Products - many protein products have been created by transferring genes into bacterium or yeast, growing it in culture and producing large quantities D. Forensics - blood or tissue left at scene can be tested using DNA sequencing. RFLP’s are used to compare DNA samples from suspects and victims yielding a DNA fingerprint. The probability that two people have the same fingerprint is very small Practical Applications E. Environmental - microorganisms can be genetically engineered to extact heavy metals, clean oil spills F. Agriculture - DNA technology is used to make vaccines and growth hormones and even transgenic organisms (organisms whose genomes carry genes from another species *genes for human proteins can be inserted into animals to make them express and produce these proteins Ethical concerns *What happens if we “create” deadly pathogens by accident? *What if genetically modified food is hazardous to people? *What if genetically modified crops hybridize with wild plants (like weeds) and create “superweeds” that are hard to control?