8 - Molecular Techniques II Electrophoresis PCR Sequencing...

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Molecular Techniques II • Electrophoresis • PCR • Sequencing Electrophoresis Separation of charged molecules based on migration in an electric field. – Separation depends on: • Electric force • Mass and shape of molecule • Matrix – Separations are performed usually in gels. • Gels act as molecular sieves • Protein gels are most commonly made from acrylamide • DNA gels are most commonly made from agarose
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electrophoresis intercalation of ethidium bromide or Sybr Safe - DNA + - + wells + - agarose gel electrophoresis Rate of DNA migration is inversely proportional to the log of MW
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Example of DNA Standards (aka ladder) known MW linear DNA always run on gel along with unknown samples PCR The Polymerase Chain Reaction (PCR) provides an extremely sensitive and rapid means of amplifying relatively large quantities of a specific DNA sequence First described in 1985, Nobel Prize for Kary Mullis in 1993. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that live in hot springs. The primary materials, or reagents, used in PCR are: 1. DNA nucleotides, the building blocks for the new DNA 2. Template DNA, the DNA sequence that you want to amplify 3. Primers, which hybridize to sequences surrounding area to be amplified 4. DNA polymerase, a heat stable enzyme that carries out the synthesis of new DNA
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Denaturation – enzymes (e.g., helicases) "unzip" or unwind the two DNA chains of the double helix. Use ATP as energy source to break bonds Annealing of primers - DNA polymerases, cannot copy a chain of DNA without a short sequence of nucleotides with a free 3’OH to "prime" the process. So the cell has another enzyme called a primase that actually makes the first few nucleotides of the copy. This stretch of RNA or DNA is called a primer. Extension - once the primer is made, a DNA polymerase can take over making the rest of the new chain. DNA polymerases work in 5’ to 3’ direction to synthesize new strand Note that a cellular supply of nucleotides is needed to act as the building block for the new DNA strand. http://nobelprize.org/educational_games/medicine/dna/a/replication/replication_ani.html Can we mimic this process in a tube? PCR uses same steps: Denaturation - instead of enzymes, heat (94°C) is used to denature template strand Annealing of primers – instead of RNA primers, synthetic DNA primers of about 20-30 nucleotides are used – temperature around 50-60°C : Extension – uses heat stable polymerase like Taq at around 72°C . – Note: DNA polymerases require Mg
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This note was uploaded on 03/31/2010 for the course BIMM bimm 100 taught by Professor Dr.sato during the Winter '08 term at UCSD.

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8 - Molecular Techniques II Electrophoresis PCR Sequencing...

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