1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.

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Transcript of 1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.

Page 1: 1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.

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PRIMER DESIGN

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Developed by Kary Mullis

Few DNA strands to millions of copies

Repeated heated and cooling

What is polymerase?What is chain reaction?

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'My God, you could use

this to isolate a fragment

of DNA from a complex

piece of DNA, from its

context.

KARY MULLIS

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Making a PCR reaction

•Taq DNA polymerase     •dNTP mixture•PCR buffer (salts, loading dye, etc)•MgCl2•ddH2O•DNA template•Primers

http://www1.qiagen.com/Products/Pcr/TaqSystem/TaqPcrCore.aspx

http://www-che.syr.edu/faculty/boddy_group/pages/thermocycler.jpg

http://upload.wikimedia.org/wikipedia/commons/8/81/PCR_tubes.png

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Primer Design

PRIMER LENGTHOptimal length – 18 – 22 bp

MELTING TEMP52 – 58 CGC content determines temp

NO SELF COMPLEMENTINGFormation of hairloop structuresIf 3’ ends complement, form primer dimers

AVOID C OR G RUNSHarder to separate

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Sample QPCR Readout

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LIGASE CHAIN REACTION

BACTERIAL

CLONING

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LACKS SELF - CORRECTION

HIGH SENSITIVITY

SHORT DNA

SEQUENCES

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