PCR Assisted Biochemistry Farzad Alemi Biochemistry 118 March 7 2000 PCR Assisted Biochemistry History Background Parameters of successful reaction Practical example Applications Future History Conceived by Kary Mullis while driving on the California freeways 1985 Received Nobel Prize for technique Since then PCR patent sold for 300 million highest amount ever for a patent Basically a method of DNA amplification Technology has wide applications to fields of Biology Biochemistry Forensics Archaeology Background ELEMENTS OF A PCR REACTION DNA Primers Enzyme Nucleotides Thermocycler Reaction Overview Exponential Amplification of DNA Original DNA After Cycle 1 After Cycle 2 After Cycle 3 After N cycles amount of target DNA is 2N 2N TAQ polymerase optimum at 72 C DNA Need to know at least the beginning and end of DNA sequence These flanking regions have to be unique to strand interested in amplifying Region of interest can be present in as little as one copy Enough DNA in 0 1 microliter of human saliva to use PCR Enzyme DNA polymerase from Thermus aquaticus Yellowstone Alternatives Thermococcus litoralis Pyrococcus furiosus Thermocycler Primers and Design Primer sequence Length 20 30 base pairs long 1 4N 50 15 G C Avoid motifs and poly N sequences Avoid inverted repeating sequences Two primers should have little complementarity 3 end of primer should be G C Melting Temperature Tm number of A T residues x 2 C number of G C residues x 4 C Both primers should have comparable melting temperatures Annealing temperature is about 5 C lower than melting temp 1 ccgtaacgga ggatgttttt cagaatgtgg ttgggattga tggatgggag gtagaacgag 61 ttcgagttga aaaggttttg tgtgccatgt gaaaaggtta gcatctatta cgtagacgag 121 agaaattcat tggaaatttg agaaggagat tgagcataat gaaacttgtt ttggaaaaat 181 atgttgttat taatgtggag gtgggcaaga atgagaataa tcagtagcaa tgaggtgtca 241 ataatttgat actgtctaca tggaagacgg cgaccagagc catggaagtc agaatgaaaa 301 atgataaatg tgaaaacatt ctagagaaga aatgaatacg cgaaggcccg tggtgggtga 361 tgacatgatg tgatttctgc ccagtgctct gaatgtcaaa gtgaagaaat tcaatgaagg 421 acgggtaaac ggcgggagta actatgactc tcttaaggta gccaaatgca tagtcatcta 481 attagtgacg ttcatgaatg gatgaacgag attcccactg tccctaccta ctatccagcg 541 aaaccacagc caaggtaacg ggcttggtgg aatccgcggg gaaagaagac cctgttgagc 601 ttgactctag tctggcacgg tgaagagcca tgagaagtgt agaataagtg ggaggcccct 661 gggcccccct gcccagcaag gggacagagt ggggcaaggc cagaggtgaa ataccactac 721 tctgattgtt tattcactga cccgtgaggt gccccaaggg gctcttgctt ctggcgccga 781 gtgcccggcc acatgcacat gccaaattgt aaagaccatc gat Sequence GC Content Position Degeneracy 3 GC 3 Degeneracy Tm Location http bibiserv techfak uni bielefeld de genefisher Forward Primer Data Reverse Primer Data GATTGATGGATGGGAGGTA CTGTGGTTTCGCTGGA 47 34 0 50 0 52 7996 34 56 533 0 50 0 51 349 533 Applications Forensics assessment reassessment of crimes Archaeology determine gene sequences of ancient organisms rethinking the past human origins Molecular Biology cloning genes RT PCR Amplification of DNA from tissues PCR on a Chip Uses Reaction complete in 2 20 minutes Extremely portable Fluorescence PCR Uses Identification purposes Real Time PCR Uses Portable means to diagnose bacteria Military medical and municipal applications Fast Results in less than seven minutes Acknowledgements Professor Brutlag Gene Fisher http bibiserv techfak uni bielefeld de genefisher Molecular Biology Techniques Manual Third Edition Edited by Coyne V et al PCR Detection of Bacteria in Seven Minutes Phillip Belgrader Science April 1999
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