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U-M BIOLOGY 172 - Begin Molecular Biology
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BIO 172 1st Edition Lecture 16 Outline of Last Lecture I. PhotosynthesisII. Molecular Biology (PCR)Outline of Current Lecture I. Molecular BiologyCurrent LectureMolecular Biology:PCR components . . . Heat Stable DNA Polymerase - Must be heat resistant because the way PCR breaks the double stranded template into a single stranded template is through heat and breaking the hydrogen bonds.Primers – DNA primers used to extend simply for the DNA polymerase to do its job. Primers can be ordered from a company.Template DNA – double stranded, and then heated so it will become two single template DNA strands.Many dNTPs Buffer PCR has repeated cycles of Denaturation, Annealing, and Extension. PCR stands for Polymerase Chain Reaction.Denaturation: Double stranded template DNA is heated, which breaks Hydrogen bonds and separates the strands.Annealing: The solution containing the single strands is cooled, then the two primers ANNEAL totheir complementary sequence on the template strand. Hydrogen bonds form between the primer and the single strands.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Extension: DNA polymerase synthesizes new DNA strands by extending the primers. After DNA polymerase copies each strand, the product becomes the new template strand!Each round of PCR multiplies how many copies there are of the template DNA.Uses for PCR: forensic analysis. Then someone who commits a crime has their DNA info on file; and later in life if they commit another crime, that person is quickly identifiable.Gel Electrophoresis: Gel electrophoresis: Agarose Gels… melt and then use a pipette to put in dye. Then the Agarose forms a mesh so that the DNA can move through based on size (smaller migrate more quickly, they move further down the cell in the same amount of time)Even though a longer fragment holds a more negative charge, the longer fragment still takes a longer time to work their way through the mesh!Where would a primer with the sequence 5’ AATCG 3’ bind to a target DNA with the total sequence of 5’ TTTAATCGTTTACGATTGGCCT 3’ ?ANSWER: at 5’ CGATT 3’ because the 3’ and 5’ end are switched, so look for the opposite. No Uracil in this because there’s not mRNA.Southern and Northern Blotting help detect a specific DNA or RNA sequence!Southern Blot detects DNA. May want to know, out of many fragments, which band has that exact sequence? The DNA is separated out by size, and then set the gel on a surface that has fluid underneath. Then a membrane placed on top of the gel… the fluid moves through both and then the membrane has the imprint of the gel on it!!Transfer of DNA to the membrane, same arrangement as it was on the gel. Once a probe comes in, the probe will base pair with the band it is complementary to!Northern blot detects RNA. RNA is already single stranded, so you could target one gene for having the most expression. Plasmids occur naturally in Bacteria: Like how PCR grows many copies of certain DNA templates,bacteria can grow many copies of plasmids. The small, circular molecules of DNA are distinct from the chromosome in the plasmid.Cloning Vector: 3 key partsORIGIN of replication; Antibiotic RESISTANCE gene; CLONING site to insert foreign DNA.The above picture is a Cloning Vector.For Amp and Tet resistance in this bacteria, two Restriction Sites would allow this plasmid to be selected for and replicated within the bacteria… So a combination of the side Hind3 and NdeI are the best choices. This is because Hind3 begins before any of the amp or tet start, so it does not cut them off. Then NdeI also does not cut off part of amp or tet.Sticky Ends and Blunt Ends: A restriction enzyme recognition site is usually a Palindrome where it reads the same from front to back, or backwards to forwards.ECO r1 restriction site. Then you have sticky ends when cut. Next complementary ends on each side of the gene are complementary to where each side of the gene was cut.This diagram is a great representation of each partof a cloning


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U-M BIOLOGY 172 - Begin Molecular Biology

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