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Lecture 9

Lecture 9 - Select Genetic Technologies

4 Pages

Course Code
Biology 2581B
Jim Karagiannis

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LECTURE 8: SELECT GENETIC TECHNOLOGIES Key Concepts 1. Basic molecular technologies Major Milestones • • 1953 – discovery of DNAstructure • 1983 – PCR, Kary Mullis o James Watson and Francis Crick • Mid 1980’s – RNAediting, simultaneously by several o Maurice Hugh Frederick Wilkins groups o Rosalind Franklin (her data was used by • Early 1990’s – micro RNA Watson and Crick) • 1976 – viral, 1995 – bacterial & eukaryotic, 2003 human • 1940/50 – transposable elements, Barbara McClintock genome • Early 1970’s – restriction enzymes,Arber, Nathans, Smith• 1995 – micro arrays • 1975 – Southern Blots, Edwin Southern • 2000 and up – new sequencing technologies, digital PCR • 1977 – Sanger DNAsequencing, Frederick Sanger large scale screening methodologies • 1978 – site specific mutagenesis, Michael Smith Restriction Enzymes • Restriction enzymes cut DNAmolecules at specific locations to produce restriction fragments with either blunt or sticky ends • Restriction enzymes have o Specific recognition sequences o Can differ in length o Result in specific cutting patterns • Fragments fit only back together if they have o Same type of ends o Matching sequences • Blunt ends can be ligated together • Enzymes have different recognition sites but they generate the same overhangs • The number of base pairs in a recognition site determines the average distance between sites in a genome and thus, the size of fragments produced • Length of recognition sequences o 4 bp will cut every every 4 = 256 bp 6 o 6 bp will cut every 4 = 4096 bp = 4.1 kb o 8 bp will cut every 4 = 65.5 kb • Human genome = 3 billion base pairs o Average length 256 bp = 12,000,000 fragments o Average length 4.1 kb = 700,000 fragments o Average length 65.5 kb = 46,000 fragments • So depending on if you want a specific average fragment length or a specific number of fragments: o Different enzyme o Different length of time for the digest  Complete versus incomplete digests Gel Electrophoresis • DNAfragments are separated by size • Agarose gel • Small fragments migrate faster than larger ones • DNAis negatively charged • Different types of gels separate different-sized DNAmolecules – gels can be composed of two different types of chemical matrices: polyacrylamide and agarose. Polyacrylamide, which has smaller pores, can separate DNAmolecules in the range of 10–500 nucleotides in length; agarose separates only larger molecules Restriction Maps • Restriction maps show the relative order and distances between multiple restriction sites, which act as landmarks along a DNAmolecule • Figure shows how a process of elimination allows you to infer the arrangement of restrictions sites consistent with the results of three sets of digestions using either of two enzymes alone or both enzymes simultaneously • How to infer a restriction map from the sizes of restriction fragments produced by two restriction enzymes 1. Divide a purified preparation of a cloned DNAinto three aliquots; expose the first aliquot to EcoRI, the second to BamHI, and the third to both enzymes 2. Separate by gel electrophoresis the restriction fragments that result from each digestion and determine their sizes in relation to defined marker 3. Use a process of elimination to derive the only arrangement that can account for the results obtained with all three samples Recombinant DNA • Vector – self-replicating DNAmolecule that can be used to transfer DNAbetween host cells and which presence can be detected • Plasmid – extra chromosomal DNAoriginally found in bacterial species • Cut DNAof interest • Cut plasmid DNA • Both are mixed together in the presence of the enzyme ligase, which sutures them to each other to form circular recombinant DNA
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