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Chapter 13: The Molecular Basis of Inheritance Concept 13.4: Understanding DNA structure and replication makes genetic engineering possible Overview ● Knowledge that two strands of DNA are complements of each other acts as a fundamental in modern genetics and genetic research ○ It is the basis upon nucleic acid hybridization, that the base pairing of one strand of nucleic acid to a complementary sequence on another strand, is built ○ Nucleic acid hybridization is the foundation for most techniques used for genetic engineering, the direct manipulation of of genes for practical purposes Genetic Engineering has drastically changed the fields of medicine, forensics, agriculture, and simple biological research DNA Cloning: Making Multiple Copies of a Gene or Other DNA Segment ◉ Naturally occurring DNA molecules are very long, and a single molecule usually carries many genes. ◉ To work directly with specific genes, scientists have developed methods for preparing well-defined segments of DNA in multiple identical copies, a process called DNA cloning. ○ One common approach uses bacteria, most often E. coli, which has large circular molecules of DNA. ■ To clone the DNA, researchers first obtain a plasmid, a small circular DNA molecule that replicates separately from the bacterial chromosome, and insert DNA from another source into it, resulting in recombinant DNA. ■ The new plasmid is then returned to a bacterial cell, creating recombinant bacterium. ■ This single cell reproduces through repeated cell divisions to form a population of genetically identical cells. Applications of Gene Cloning ◉ Gene cloning is useful for two basic purposes- to amplify, or make copies of a gene, and to produce a protein product. ○ Organisms can be given new, metabolic traits, such as pest resistance. ○ Proteins with medical uses can be harvested in large quantities from cultures of bacteria carrying the cloned gene from the protein. Using Restriction Enzymes to Make Recombinant DNA ◉ Overview ○ Genetic cloning and engineering uses restriction enzymes to recognize and cut DNA at specific locations called restriction sites. ■ This was discovered while researching bacteria, which protects it by cutting foreign DNA, but it protects itself from its own enzymes by adding methyl groups to adenines and cytosines in recognized sequences. ○ When DNA is cut, it creates a set of segments called restriction fragments. ■ Fragments are identical when cut with the same enzyme. ● Most restriction sites are symmetric (nucleotides are the same on each strand, same in 5’ → 3’) ● Most commonly used is 4’ → 8’, occurs many times Over DNA molecule ● This restriction enzyme recognizes 6 base pair sequences making staggered cuts in the sugar phosphate backbones ● Results in sticky ends, any fragments with complementary ends may base- pair. ○ the resulting ligated product will be recombinant DNA if the fragments come from different molecules ● ● ● ● When DNA is cut, it creates a set of segments called restriction fragments. Fragments are identical if cut with the same enzyme. Seeing these requires electrophoresis- polymer separates nucleic acids by size, electric charge and other physical properties in the electric field. Sugar phosphate backbones in DNA strands result in double stranded fragments, which have a single stranded end, or sticky end forming temporary hydrogen bonds with molecules cut by the same enzyme. In gene cloning, DNA molecules that are to be joined together are a cloning vector- a DNA molecule able to carry foreign DNA to a host cell for replication, often a bacterial plasmid with one restriction site recognized by its specific restriction enzyme Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) and Its Use in Cloning ◉ When scientists need to analyze DNA with only a small sample size, they use polymerase chain reaction (PCR) to amplify and copy genetic material ○ PCR has the capability to make millions (or even billions) of copies of small sections of DNA Number of copies equals 2^n, were n is the number of cycles A target sequence can make up as little as 0.001% of the total DNA in a sample ○ PCR cannot replace gene cloning because of its error rate as samples become larger ○ PCR has been used to amplify DNA from 40,000 year-old frozen woolly mammoth, small amounts of DNA at crime scenes, and single embryonic cells for unique and fast prenatal treatment for genetic disorders Essentially, there are three parts... Denaturation Annhelling The DNA is heated to The DNA is cooled to allow separate the two strands for short-sequence primers to attach to anneal (base pair) with its complementary sequence. The primers isolate the target sequence for amplification. Extention Temperature is raised once again to allow Taq polymerase to attach to each priming site and extend (synthesize) a new DNA strand. The Secret To Polymerase Chain Reaction ◉ If a normal DNA polymerase were used, both the protein and DNA would be denatured in the heating at the beginning of the cycle ○ This means the protein would have to be replaced after every cycle ○ The key to PCR being an automatic procedure is a the discovery of a heat-stable polymerase called Taq polymerase ■ Named after the bacteria Thermus aquaticus which lives in hot springs ■ This bacteria can live in temperatures up to 95 degrees celsius DNA Sequencing ◉ DNA sequencing- the process of exploiting the principle of complementary base pairing to determine the gene’s complete nucleotide sequence ◉ Recent developments have lead to sequencing techniques that are rapid and inexpensive. ○ Now, a single template strand can be immobilized and DNA polymerase, among other reagents, are added to allow sequencing by synthesis of the complementary strand, one nucleotide at a time. ○ Electronic monitors are used to identify which of the four nucleotides are being added, determining the sequence. Bibliography Reece, Jane B, and Neil A. Campbell. Campbell Biology. Boston: Benjamin Cummings / Pearson, 2011. Print. “ Thanks! Any questions?