Stanford BIO 118 - PCR Assisted Biochemistry

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PCR Assisted BiochemistryFarzad AlemiBiochemistry 118March 7, 2000PCR Assisted Biochemistry• History• Background• Parameters of successful reaction• Practical example• Applications• FutureHistory• Conceived by Kary Mullis while driving onthe California freeways (1985)• Received Nobel Prize for technique• Since then, PCR patent sold for $300million -- highest amount ever for a patent• Basically, a method of DNA amplification• Technology has wide applications to fieldsof• Biology•Biochemistry• Forensics• ArchaeologyBackground• DNA• Primers• Enzyme• Nucleotides• ThermocyclerELEMENTS OF A PCR REACTION:Reaction Overview: Exponential Amplification of DNAOriginal DNAAfter Cycle 1After Cycle 2After N cycles, amount of target DNA is 2N-2NAfter Cycle 3TAQ polymerase optimum at 72° CDNA•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 ThermocyclerPrimers and DesignPrimer sequence:Length: 20-30 base pairs long (1/4N)50% ± 15% G-CAvoid motifs and poly-N sequencesAvoid inverted repeating sequencesTwo primers should have little complementarity3’ end of primer should be G/CMelting TemperatureTm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]Both primers should have comparable melting temperaturesAnnealing temperature is about 5° C lower than melting tempForward Primer Data Reverse Primer DataSequence GATTGATGGATGGGAGGTA CTGTGGTTTCGCTGGAGC Content 47 56Position 34 533Degeneracy 0 03' GC 50 503' Degeneracy 0 0Tm 52.7996 51.349Location 34 5331 ccgtaacgga ggatgttttt cagaatgtgg ttgggattga tggatgggag gtagaacgag61 ttcgagttga aaaggttttg tgtgccatgt gaaaaggtta gcatctatta cgtagacgag121 agaaattcat tggaaatttg agaaggagat tgagcataat gaaacttgtt ttggaaaaat181 atgttgttat taatgtggag gtgggcaaga atgagaataa tcagtagcaa tgaggtgtca241 ataatttgat actgtctaca tggaagacgg cgaccagagc catggaagtc agaatgaaaa301 atgataaatg tgaaaacatt ctagagaaga aatgaatacg cgaaggcccg tggtgggtga361 tgacatgatg tgatttctgc ccagtgctct gaatgtcaaa gtgaagaaat tcaatgaagg421 acgggtaaac ggcgggagta actatgactc tcttaaggta gccaaatgca tagtcatcta481 attagtgacg ttcatgaatg gatgaacgag attcccactg tccctaccta ctatccagcg541 aaaccacagc caaggtaacg ggcttggtgg aatccgcggg gaaagaagac cctgttgagc601 ttgactctag tctggcacgg tgaagagcca tgagaagtgt agaataagtg ggaggcccct661 gggcccccct gcccagcaag gggacagagt ggggcaaggc cagaggtgaa ataccactac721 tctgattgtt tattcactga cccgtgaggt gccccaaggg gctcttgctt ctggcgccga781 gtgcccggcc acatgcacat gccaaattgt aaagaccatc gathttp://bibiserv.techfak.uni-bielefeld.de/ genefisher/ApplicationsForensics• assessment/reassessment of crimesArchaeology • determine gene sequences of ancient organisms • rethinking the past, human originsMolecular Biology • cloning genes • RT-PCR • Amplification of DNA from tissuesPCR on a ChipUses: Reaction complete in 2-20 minutesExtremely portableFluorescence PCRUses: Identification purposesReal-Time PCRUses: • Portable means to diagnose bacteria• Military, medical, and municipal applications• Fast: Results in less than seven minutesAcknowledgementsProfessor BrutlagGene 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


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Stanford BIO 118 - PCR Assisted Biochemistry

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