Lec39s - DNA Technology Part II. Chap. 20 th ed.: 382-386 6...

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DNA Technology Part II. Chap. 20 6 th ed.: 382-386 7 th ed.: 391-398 8 th ed.: 403-409 TODAY: • PCR • SNPs/RFLPs • DNA Sequencing
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PCR: polymerase chain reaction Makes many copies of a piece of DNA in vitro (without cells)
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Advantages of PCR - fast - need very little starting material - not limited by restriction sites in DNA
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For PCR, need only : -DNA template (contains sequence to be copied) -DNA polymerase (heat-stable!) -dNTP’s and. ...
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2 synthetic DNA primers; complementary to ends of desired sequence on opposite strands. Need these in excess over template.
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PCR: repeat a 3-step cycle many times 1.Heat : to denature (separate strands). 2.Cool : primers bind template strands 3.Extend : DNA poly- merase makes a new strand, starting with primer
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Targeted sequence Heat to denature Cool (primers bind) A B
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Extend new strands ~ End of Cycle I ~ A B
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Cycle 2 : primers can bind either to old strands or new strands (made in Cycle 1). “Primer A” can bind to the strand made by “Primer B” in Cycle 1- and vice versa.
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A A B A Primer A can bind either to the template strand or to a strand just made by Primer B ~ OR ~ start of Cycle 2
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strand (made in Cycle I), it runs off the end during extension. The product is the desired fragment! B
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Lec39s - DNA Technology Part II. Chap. 20 th ed.: 382-386 6...

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