DNA Technology. B. Using Bacteria to Clone Genes: Overview:

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1 DNA Technology A. Basic Vocabulary: is DNA from 2 different sources that is combined. is the direct manipulation of genes for practical purposes. literally means or in a test tube or flask. is the manipulation of organisms to perform practical tasks or provide useful products. For example: using microbes to make wine or cheese; selective breeding of livestock or crops; moving genes from a mammal to a plant. is making well-defined gene-sized pieces of DNA in multiple identical copies. B. Using Bacteria to Clone Genes: Overview: are enzymes that occur naturally in bacteria that protect the bacteria against intruding from other organisms. They work by foreign DNA at points along the DNA strand. A specific restriction enzyme recognizes specific short sequences in DNA called a. Most restriction sites are and nucleotides in length. The restriction enzyme has to cut and bonds of both strands and the cuts produce fragments of DNA with stranded DNA called. Because one type of restriction enzyme always cuts at the same restriction site, the same are

2 always produced. And since the target sequence may occur many times in a DNA molecule, this produces many fragments. The sticky ends of the fragments then easily form bonds with their base pairs. This occurrence can bring about a DNA molecule if it is from source. Then seals the strands together, forming covalent phosphodiester bonds. A Closer Look: The plasmid that will be cut with a restriction enzyme and that can carry a piece of DNA into a cell and replicate there is called a. bacterial plasmids are the most commonly used cloning vectors.

3 Step 5 on the figure above is the most difficult part because we will have cloned many human DNA fragments. To distinguish the colony containing the gene of interest we can either A) look for the itself or B) look for its product. To look for the gene directly, a stranded nucleic acid can be used in a method called nucleic acid. If we know the nucleotide sequence of our gene, we can make a radioactive probe to it. Then by (with heat or chemicals) the of the newly cloned DNA, the radioactive probe can the correct clone by hydrogen bonding to its single-stranded complement. Now that the desired gene is found, the cells containing the gene of interest are cultured in large amounts.

4 C. Prokaryotic/ Eukaryotic Problems: There are some problems in getting a to function with DNA in it. One problem is that while prokaryotes lack, eukaryotes have these segments of non-coding DNA. Scientists must make eukaryotic genes that lack introns. This is done by allowing a cell to undergo transcription and make, then allowing spliceosome to remove. The mrna is then added to a solution of transcriptase which creates a strand of, minus the introns. This intron free DNA is called DNA (cdna) and can now be inserted into a vector. To avoid the prokaryote-eukaryote incompatibility, scientists sometimes use. Yeast are -celled that grow as easily as bacteria, have, and are. Also, scientists have made that are vectors containing an origin of replication, a, 2, and most importantly DNA. These artificial chromosome vectors are much longer than plasmid vectors enabling long pieces of DNA to be. Another reason to use eukaryotic host cells for expressing a cloned gene is that many proteins are after translation by the addition of a or. Therefore, host cells from an animal or plant cell culture may be necessary. Also, eukaryotic cells can receive brief impulses that create in the plasma membrane. This process is called and the temporary holes then allow to enter. Similarly, scientists can also DNA into a eukaryotic cell using microscopic. D. PCR: is used to clone scanty or DNA. This technique is used to clone of any piece of DNA (in a few hours). PCR starts with a special kind of, a supply of, single stranded DNA, and the DNA to be cloned in a test tube. The DNA to be cloned is then to separate the

5 strands, then the hydrogen bond to each strand, and adds nucleotides in its direction. The solution is then heated again and the cycle repeats again and again for about cycles. Because the DNA cloned by PCR can be in small amounts or partially degraded, it has many applications. Including, in murder trials when only a small amount of DNA is left at the scene of the crime and it s used to make many copies of DNA from a 40,000-year-old frozen. E. Analyzing Cloned DNA: Now that we understand a few ways to clone DNA, lets see how we analyze the DNA (aside from nucleic acid hybridization). Most methods make use of. This technique separates or on the basis of and charge. Gel electrophoresis sorts a mixture of DNA into along the gel. analysis is often used to compare different alleles of a gene or DNA of different individuals or species. This technique involves treating the DNA

6 molecules in question with enzyme. Then doing which yields different banding patterns called (RFLPs) when homologous chromosomes vary in length of fragments due to different cuts by restriction enzymes. The cuts vary because homologous chromosomes can be different. Then the fragments are further analyzed by another technique called. This involves putting the gel from restriction fragment analysis in an solution and putting blotting paper on top of it. This transfers the DNA from the to the and denatures it. Then the paper blot is exposed to a single-stranded radioactive nucleic acid that forms complementary base pairs to the DNA on the blotting paper. The analysis is then completed by exposing the radioactive probes to which yields specific DNA bands. F. Mapping the Entire Genome: In 1990 the officially began to find the precise location of all an organism s and as well. In addition to mapping human DNA, researchers are mapping the genomes of,,, and. These DNA sequences can then be compared between organisms and confirm connections between even distantly related organisms.

7 Moreover, by comparing the completed genomes of,, and scientists now have support for the theory that these 3 represent the or superkingdoms of life. G. Applications of DNA Technology: Biotechnology is making enormous contributions to medicine, especially in diagnosing. Hundreds of human genetic disorders have been identified the onset of symptoms. In addition, of potentially harmful alleles can now be identified as well. Human may someday enable scientists to genetic disorders by a defective allele with a functional one using DNA techniques. For gene therapy of somatic cells to be permanent, the cells that receive the normal allele must be ones that throughout the patient s life. The new allele could be inserted into the somatic cells of the tissue affected by the disorder in a child or adult or even the line cells (produce gametes) or cells. Of course there is much controversy over whether gene therapy is ethical and some worry this may lead to the practice of - an effort to control the genetic makeup of human populations. DNA technology has also created many pharmaceutical products, mostly. As well as hormones like insulin for diabetics, to help correct a form of dwarfism, and to dissolve blood clots and reduce the risks of heart attacks. The problem with these genetically engineered products is developmental costs are high and the market isn t very broad, so the products are. production is also changing with the help of biotechnology. Ordinarily viruses or bacteria were killed or weakened by the use of or. Now techniques are a safer alternative to creating mutants that do not cause disease. DNA technology is also being used in crime scenes because every individual s DNA is, except. analysis by Southern blotting/radioactive probes is a common method used to detect similarities and differences in DNA samples and only requires tiny amounts of blood or tissue. Usually forensic scientists only test a portions in DNA that are highly variable from person to person. This is called a. Many bacteria can extract heavy (Cu, Pb, Ni) from the environment which is important as ores get depleted. In addition, microbes can degrade some compounds released after an. Also, some bacteria organic compounds into nontoxic forms helping out sewage and water treatment plants.

8 organisms- organisms that contain genes from another species, have also been developed by injecting foreign DNA into the nuclei of or early embryos. have been treated with products made by recombinant DNA methods. For example, some have been injected with made by E. coli to raise milk production. BGH also improves weight gain in. Plants have been much easier to genetically engineer because with most plants an adult can be grown from cell. The vector most commonly used to move genes into plants is the plasmid from the Agrobacterium tumefaciens, so not E. coli! Normally this bacterium infects plants and causes called. This crown gall causing plasmid is called the (tumor inducing) plasmid. When scientists wish to insert a new gene into a plant by use of the T i plasmid, they the genes that cause the disease and replace it with foreign DNA. The only drawback of using the T i plasmid as a vector is it only infects plants like beans, roses, peas, sunflowers, oaks and maples. All these plants are dicots because they have seed leaves in addition to other similarities we will learn about later. So plants like wheat, corn, rice, barley, orchids, palms, lilies, and grasses cannot be genetically engineered with the aide of Agrobacterium. The monocots must rely on and DNA needles to get DNA into these plants. Many of the genetically engineered plants have genes for resistance. This helps farmers to control and not destroy the crops. Also, many crop plants are being engineered to resist, enabling farmers to reduce the need to apply chemical. Also, many fruits are being genetically engineered to contain genes that retard. This is done by blocking the production of the ripening so plants only produce less of these. Plants are also being genetically engineered to. Recall that N 2 gas is fixed by bacteria called living on the roots of plants like beans, peas, and alfalfa. Then this nitrogen cycles its way through different microbes living in the soil, eventually being converted to the usable form by all plants called. All life needs nitrogen for and. By genetically engineering plants to fix, less nitrogen fertilizers will have to be applied and consequently, less to the environment and lower costs to farmers.

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