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Brad Porter

Lecture: Introduction to PCR & Analysis of Gene Expression Using RT-PCR . Fri, June 15, 2007 11:00 – 11:50 AM  . Brad Porter . Briefly, what is PCR?. Primers anneal to single stranded DNA ~55 °C. Thermostable TAQ polymerase extends primers at ~72°C. 3’. 5’. 3’. 5’.

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Brad Porter

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  1. Lecture: Introduction to PCR & Analysis of Gene Expression Using RT-PCR Fri, June 15, 2007 11:00 – 11:50 AM  Brad Porter

  2. Briefly, what is PCR?

  3. Primers anneal to single stranded DNA ~55°C Thermostable TAQ polymerase extends primers at ~72°C 3’ 5’ 3’ 5’ Target DNA is doubled. Cycle is then repeated. Polymerase Chain Reaction Target DNA DNA Denatures at 94°C http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

  4. How was PCR discovered?

  5. PCR originates from DNA sequencing. So, lets first review DNA sequencing.

  6. 3’ 5’ Primer 3’ 5’ Primer Anneals & DNA Polymerase Adds Deoxynucleoside triphosphates Sequencing is performed by DNA replication 37°C Extension New DNA strand is created

  7. dNTPs (or bases) are being added, but we do not know the sequence. 3’ 3’ 3’ 5’ 5’ 5’ ATGCATGCATGC???????????????????????????????????? ATGCATGCATGC???????????????????????????????????? ATGCATGCATGC???????????????????????????????????? TACGTACGTACG 3’ 5’ Primer DNA Pol. TACGTACGTACG???????????????????????????????? 5’ 3’ Primer DNA Pol.

  8. 3’ 3’ 5’ 5’ ATGCATGCATGC???????????????????????????????????? ATGCATGCATGC???????????????????????????????????? DNA Pol. DNA Pol. A What if DNA extension could be terminated at a known nucleotide using a mixture of normal bases and termination bases TACGTACGTACGTGT 5’ A Primer By probability termination will occur at every “A” TACGTACGTACGTGT CG A 5’ Primer Normal base gets incorporated

  9. A T G dATP+ dGTP dCTP dTTP C What if four reactions were set up to stop at each nucleotide? DNA dATP dGTP+ dCTP dTTP dATP dGTP dCTP dTTP+ dATP dGTP dCTP+ dTTP

  10. 3’ 3’ 5’ 5’ ATGCATGCATGC???????????????????????????????????? ATGCATGCATGC???????????????????????????????????? DNA Pol. DNA Pol. T T TACGTACGTACG 5’ Primer TACGTACGTACG G T 5’ Primer Normal base gets incorporated

  11. 3’ 3’ 5’ 5’ ATGCATGCATGC???????????????????????????????????? ATGCATGCATGC???????????????????????????????????? DNA Pol. DNA Pol. G G TACGTACGTACGT 5’ Primer G TACGTACGTACGT TAC 5’ Primer Normal base gets incorporated

  12. 3’ 5’ ATGCATGCATGC???????????????????????????????????? DNA Pol. C TACGTACGTACGTGTA 5’ Primer

  13. TACGTACGTACG?????? 5’ TACGTACGTACG????? 5’ TACGTACGTACG??????? TACGTACGTACG??????? TACGTACGTACG??????? TACGTACGTACG??????? 5’ 5’ 5’ 5’ A A T T G G C TACGTACGTACG 5’ TACGTACGTACG??? 5’ TACGTACGTACG? 5’ TACGTACGTACG?? 5’ TACGTACGTACG???? 5’

  14. TACGTACGTACG?????? 5’ TACGTACGTACG????? 5’ TACGTACGTACG??????? 5’ A A T T G G TACGTACGTACG??? 5’ TACGTACGTACG?? 5’ C TACGTACGTACG? 5’ TACGTACGTACG 5’ Chain-termination provides sequence! 3’ TACGTACGTACG???? 5’ 5’

  15. dATP +ddATP dGTP dCTP dTTP dATP dGTP + ddGTP dCTP dTTP dATP dGTP dCTP dTTP+ ddTTP dATP dGTP dCTP+ ddCTP dTTP What causes chain termination? DideoxynucleosideTriphosphates

  16. Chain Extension 5’ DNA Polymerase Deoxynucleoside triphosphates Deoxy adenosine triphosphate (dATP) Deoxy guanosine triphosphate (dGTP) Deoxy thymidine triphosphate (dTTP) Deoxy cytidine triphosphate (dCTP) 3’ Chain Termination 5’ DNA Polymerase 3’ X Lacks a 3’ hydroxyl group. Acts as a terminator because, once incorporated, no other nucelotide can be added. Dideoxynucleoside triphosphates Dideoxy adenosine triphosphate (ddATP) Dideoxy guanosine triphosphate (ddGTP) Dideoxy thymidine triphosphate (ddTTP) Dideoxy cytidine triphosphate (ddCTP)

  17. PhD 1943 Cambridge University Nobel Prize In Chemistry 1958 Amino acid sequence of insulin Nobel Prize In Chemistry 1980 Sequenced the first genome, phage Φ-X174, by hand using a method that he developed. Frederick Sanger The Sanger Dideoxy sequencing method was the foundation for the discovery of PCR.

  18. Dideoxy sequencing, one more time. http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

  19. Ok, but what is the connection between DNA sequencing and PCR?

  20. 1983 Emeryville, California Cetus Corporation Henry Erlich was working on methods for detecting point mutations. 5’-TACGTACGTACGA*GGAGTCCGGAATG-3’ A? T? G? C?

  21. Why not do Sanger sequencing at a single base pair? ddATP ddGTP ddTTP ddCTP 5’-TACGTACGTACGA*GGAGTCCGGAATG-3’ CCTCAGGCCTTAC-5’ + Kary B. Mullis

  22. First step to a Nobel Prize: As you think, ignore obvious problems.

  23. Kary wanted to use total genomic DNA, but he forgot the primer would likely mis-pair and ruin his experiment. In “misguided puttering”, Kary kept thinking! CCTCAGGCCTTAC-5’ CCTCAGGCCTTAC-5’ CCTCAGGCCTTAC-5’ CCTCAGGCCTTAC-5’

  24. Kary and girlfriend chemist Jennifer Barnett Mendocino County

  25. What if I use two primers for confirmation? 5’-TACGTACGTACGA*GGAGTCCGGAATG-3’ CCTCAGGCCTTAC-5’ R-short F-long 5’-GAATTCTACGTACGTACGA 3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’ ddATP ddGTP ddTTP ddCTP +

  26. ddATP ddGTP ddTTP ddCTP 5’-TACGTACGTACGATGGAGTCCGGAATG-3’ ACCTCAGGCCTTAC-5’ R-short F-long 5’-GAATTCTACGTACGTACGAT 3’-ATGCATGCATGCTACCTCAGGCCTTAC-5’ + F-long R-short

  27. What about stray nucleotide triphosphates? F-long 5’-GAATTCTACGTACGTACGA 3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’ ddATP ddGTP ddTTP ddCTP dNTP 5’-TACGTACGTACGA*GGAGTCCGGAATG-3’ CCTCAGGCCTTAC-5’ R-short dNTP +

  28. I can destroy stray dNTPs with alkaline phosphatase! But, bacterial alkaline phosphatase will remain because it cannot be heat killed. It will destroy the ddNTP’s (not true).

  29. Second step to a Nobel Prize: Make up problems that do not exist and try to solve them.

  30. 5’-TACGTACGTACGA*GGAGTCCGGAATG-3’ CCTCAGGCCTTAC-5’ R-short F-long 5’-GAATTCTACGTACGTACGA 3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’ I can deplete nucleotides by adding polymerase first without ddNTP’s dNTP dNTP

  31. Third step to a Nobel Prize. Recognize PCR when you find it. Anderson Valley 1 Copy Denature and anneal primers Polymerase extension 2 Copies! DNA replicated!

  32. NOBEL PRIZE! …..not so fast

  33. Final step to a Nobel Prize: Try to get someone to listen to you.

  34. At first, people did not get it. “…no one was particularly enthusiastic about it.” 1984 annual Cetus Scientific Meeting…..”nobody seemed to be interested in my poster….” “People would glance at it and keep walking.” Then Joshua Lederberg (also a Nobel Laureate) said: “Why didn’t I think of that?”

  35. 1993 Nobel Prize

  36. So, how is PCR important to your life, right now? Many SNP’s are associated with disease. Do you have a risk allele?

  37. Let’s set up a PCR reaction and find out!

  38. GCAGCTCACCTCCAGCTTTAGTTTTC[C/T]CATGACAGTAAGTCTATTACCCTCC Risk allele Loci for Type 2 Diabetes and Triglyceride Levels

  39. First, you need to select primers for PCR • No, you do not need a computer program to select primers. • I prefer 24 bp long and end on G or C • Others prefer 20 bp long and end on A or T • Try to have at least 50% G’s +C’s to ensure reasonable annealing temperature That’s about it. Do not waste too much time selecting primers.

  40. Forward Primer Selection TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’

  41. Reverse Primer Selection TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’

  42. Amplified Fragment Will Be 230bp AGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGC

  43. Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’ Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’

  44. TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAACTAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC 5’-AGAACACCATAACCTTTCCGG CCC-3’ Before ordering: Imagine the primers annealing to the DNA Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’ AGCCACTGCACCCGACTGG-3’ Reverse Primer: 5’-GCGTG Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’

  45. Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’ Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’ Order From IDT

  46. Finish Start PCR Components H20 28.25μl 10XPCR Buffer 5.0μl 25mM MgCl2 3.0μl 4mM dNTP’s 2.5μl 10pmol Forward Primer 5.0μl (10pmol) 10pmol Reverse Primer 5.0μl (10pmol) Template DNA >104 copies of target sequence (1.0μl) TAQ Polymerase 0.25μl (5u/μl)

  47. Use thin-wall PCR tubes Use thin-wall tubes designed for PCR

  48. Old School “Perkin Elmer 2400” PCR Thermocycler Hot bonnet prevents condensation.

  49. Mineral Oil Sample If your thermocycler does not have a hot bonnet or if you will need to open the hot bonnet to remove or modify a reaction, use mineral oil.

  50. Runs on antifreeze and refrigerant Paper towels to adsorb leaking orange-colored antifreeze.

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