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Lecture 9

Lecture 9: "Select Genetic & Genomic Technologies"

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Biology 1001A
Susanne Kohalmi

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Genetics Lecture No. 9: Select Genetic & Genomic Technologies th Wednesday February 6 , 2013 Restriction Enzymes: -Restriction enzymes recognize a specific sequence of bases anywhere within the genome and then sever two covalent bonds (one in each strand) in the sugar-phosphate backbone at particular positions within or near that sequence. They have specific recognition sequences, differing in length, that result in x specific cutting patterns. Restriction sequences with length x will cut every 4 base-pairs, producing fragments of the same size. To find the amount of fragments made, divide the total number of base- pairs in the genome by the estimated length of each fragment. The greater the restriction sequence’s length, the smaller the number of fragment produced. The restriction enzyme used depends on the desired: specific average fragment length or specific number of fragments. These are achieved by using different enzymes, different digestion time lengths or complete versus incomplete digestions. Gel Electrophoresis & Recombinant DNA: -In gel electrophoresis, DNA fragments are separated by size on an agarose gel using an electric current. Smaller DNA fragments will migrate faster to the positive end of the gel (DNA is negatively-charged) than larger DNA fragments. The sizes of the bands in the unknown samples can be calibrated by comparison to size markers that have been run in the leftmost lane of the gel. Restriction digestion can be used to do mapping by first dividing a purified preparation of cloned DNA into three portions (exposing the first portion to EcoRI, the second to BamHI, and the third to both enzymes. Next the resulting fragments are separated by gel electrophoresis and their sizes in relation to defined markers are determined. Finally we use a process of elimination to derive the only arrangement that can account for the results obtained with all three samples. -A vector is any self-replicating DNA molecule that can be used to transfer DNA between host cells and whose presence can be detected. A plasmid is an extra chromosomal DNA originally found in bacterial species. We can create recombinant DNA molecules by first cutting human genomic DNA with EcoRI to produce a mixture of fragments. A plasmid vector is also cut with EcoRI at its single EcoRI recognition site. The two are mixed together in the presence of the enzyme ligase, which anneals them to each other to form circular recombinant DNA molecules. E. coli cells later transformed with the recombinant plasmids are recognized by their growth in the presence of ampicillin. Polymerase Chain Reaction: -Polymerase Chain Reaction (PCR) is a powerful tool used to isolate and make large quantities of a defined DNA fragment from a complex genome. PCR takes advantage of hybridization and synthetic oligonucleotides. Its starting material can be as small as a single cell. Because amplification is exponential, a single DNA molecule can be copied into trillions of copies in a single day. Your synthetic oligonucleotides (one forward primer and one reverse primer) must recognize the template DNA in order to amplify both strands of the target DNA region by fl
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