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DNA Technology Genetic Engineering • Alteration of genomes for medical or industrial purposes. – Can be used in bacteria, plant, and animal cells The Cloning of a Gene • Cloning – The production of identical copies through some asexual means. – All human identical twins • Gene Cloning – The production of many identical copies of a single gene. The Cloning of a Gene • Why? – Determine the difference between normal and mutated genes – Alter the phenotype of organisms • Called gene therapy in humans Recombinant DNA Technology • Contains DNA from two or more sources – Ex. human and bacteria • Vector: a piece of DNA that can be manipulated, in order to add foreign DNA to it – Plasmids: small accessory rings of DNA from bacteria that are not part of the bacterial chromosome and are capable of replicating on their own. Most common in bacteria Recombinant DNA Technology • Using Recombinant DNA Technology (Figure 24.15) – Restriction enzymes cleave (break open) a plasmid vector at specific site. Foreign DNA that is to be inserted is also cleaved with same restriction enzyme – The single-stranded, but complementary, ends of the two DNA molecules are called “sticky ends”. – Foreign DNA is inserted into plasmid DNA and “sticky ends” pair up – DNA ligase seals them together – Bacterial cells take up the recombinant plasmids. As plasmid replicates, the gene is cloned. Recombinant DNA Technology Polymerase Chain Reaction • Can create millions of copies of a segment of DNA – quickly in a test tube – No vector or host cell • It amplifies (makes copies of) a targeted DNA sequence • Requires DNA polymerase, a set of primers, and a supply of nucleotides – Primers are single stranded DNA sequences that start replication • Amount of DNA doubles with each replication Polymerase Chain Reaction • During the PCR cycle, the mixture of DNA, DNA polymerase, two primers, and nucleotides is heated to 95°C to separate the two strands of the double helix – The DNA polymerase was extracted from the bacterium Thermus aquaticus, which lives in hot springs • The temperature is lowered so the primers pair with the DNA strands • DNA polymerase copies the DNA between the primers • At the end of the second cycle there are 4 copies, after 25-30 cycles there are over 30 million copies DNA Fingerprinting • Permits identification of individuals and their relatives. • Based on differences in the sequence of DNA nucleotides between individuals. DNA Fingerprinting Medical use: identify the presence of a viral infection or mutated gene. Forensics: DNA from a crime scene can identify the suspect. Identify people: can identify the parents of a child or the identity of body remains. Biotechnology • Uses natural biological systems to create a product or to achieve an end desired by human beings. – Bacteria, plants, and animals • Transgenic organisms: organisms that have had a foreign gene inserted into them. Transgenic Bacteria • Recombinant DNA technology is used to produce transgenic bacteria. • Medical Uses: insulin, human growth hormone, hepatitis B vaccine • Agricultural Uses: bacteria that protect plants from freezing, bacteria that protect plant roots from insects. • Environmental: bacteria that degrade oil, bacteria that remove sulfur from coal Transgenic Plants • Foreign genes are introduced into plant embryos or protoplasts – When protoplasts are suspended in a liquid containing foreign DNA, an electric current makes tiny holes in the plasma membrane where genetic material can enter. • Protoplasts develop into mature plants containing and expressing the foreign DNA Transgenic Plants • Makes plants that are resistant to herbicides (soybeans), plants that secrete toxins that kill insects (cotton, corn, potato), some are both • Plant Pharming: the use of engineered plants to produce pharmaceuticals – Golden rice: designed to be grown and consumed in areas with a shortage of dietary vitamin A, a deficiency which is estimated to kill 670,000 children under the age of 5 each year. Transgenic Animals • Genes are inserted into the eggs of animals. • By hand or vortex mixing – eggs placed in a agitator with DNA and silicon-carbide needles. – The needles make holes in the eggs where the DNA can enter. – When the eggs are fertilized, the offspring are transgenic animals. • Bovine growth hormone: used to make larger fishes, cows, pigs, rabbits, and sheep Transgenic Animals • Animal pharming: the use of transgenic farm animals to produce pharmaceuticals – DNA containing the gene of interest is injected into donor eggs. – Following fertilization in the lab, the zygotes are placed in host females, where they develop – The offspring will produce the biotechnology product • Drugs in the milk of goats DNA Fingerprinting • Restriction enzymes cut DNA into fragments • Fragments are separated by gel electrophoresis, – DNA fragments migrate though a gel according to their length when an electric field is applied. – The shorter the fragment, the farther it migrates – Carried out by detecting how many times a short sequence is repeated • People differ by how many repeats they have at particular locations – PCR amplifies only particular portions of the DNA. The greater the number of repeats, the greater the amount of DNA that is amplified by PCR. The