Romans 5:17

1. Romans 5:17 17 For if by one man’s offence death reigned by one; much more they which receive abundance of grace and of the gift of righteousness shall reign in life by one, Jesus Christ.

2. Polymerase Chain Reaction Timothy G. Standish, Ph. D.

3. History • The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention • A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers • Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry

4. What PCR Can Do • PCR can be used to make many copies of any DNA that is supplied as a template • Starting with one original copy an almost infinite number of copies can be made using PCR • “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis • Defective genes can be amplified to diagnose any number of illnesses • Genes from pathogens can be amplified to identify them (i.e., HIV) • Amplified fragments can act as genetic fingerprints

5. How PCR Works • PCR is an artificial way of doing DNA replication • Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times • As in replication, PCR involves: • Melting DNA • Priming • Polymerization

6. Initiation - Forming the Replication Eye Origin of Replication 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’

7. Extension - The Replication Fork 3’ 5’ 5’ 3’ 3’ 5’ 3’ Primase 5’ Single-strand binding proteins Lagging Strand 5’ 5’ 3’ 5’ RNA Primers DNA Polymerase 5’ 3’ Helicase Leading Strand 5’ 3’ Okazaki fragment

8. Functions And Their Associated Enzymes Function Enzyme • Ligase • Melting DNA • Helicase • SSB Proteins • Topisomerase • Polymerizing DNA • DNA Polymerase • Providing primer • Primase • Joining nicks

9. Components of a PCR Reaction • Buffer (containing Mg++) • Template DNA • 2 Primers that flank the fragment of DNA to be amplified • dNTPs • Taq DNA Polymerase (or another thermally stable DNA polymerase)

10. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers 72 oC Temperature 50 50 oC 0 T i m e 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’

11. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’

12. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ Heat 5’ 3’

13. PCR Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’

14. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ Heat Heat 5’

15. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’

16. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ Heat Heat

17. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’

18. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’

19. DNA Between The Primers Doubles With Each Thermal Cycle Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6

20. More Cycles = More DNA Number of cycles 0 10 15 20 25 30 Size Marker

21. Theoretical Yield Of PCR Theoretical yield = 2n x y Where y = the starting number of copies and n = the number of thermal cycles If you start with 100 copies, how many copies are made in 30 cycles? 2nx y = 230x 100 = 1,073,741,824 x 100 = 107,374,182,400

22. How The Functions Of Replication Are Achieved During PCR Function PCR • N/A as fragments are short • Melting DNA • Heat • Polymerizing DNA • Taq DNA Polymerase • Providing primer • Primers are added to the reaction mix • Joining nicks

23. Using PCR To Fingerprint DNA • Because PCR can amplify any targeted segment of DNA (at least in theory) highly variable regions of the human DNA can be amplified and compared • The simplest variations in DNA to detect are variations in length • Thus polymorphic regions of DNA in which Variable Number Tandem Repeats (VNTRs) occur are excellent targets for PCR based DNA fingerprinting • pMCT118, the VNTR we will target, is a 16 bp repeat which varies between 14 and 40 copies on chromosome 1

24. pMCT118 …GCTCAGTGTCAGCCCA 1 AGG-AAGACAGACCAC 2 AGGCAAGGAGGACCAC 3 CGGAAAGGAAGACCAC 4 CGGAAAGGAAGACCAC 5 CGGAAAGGAAGACCAC 6 AGGCAAGGAGGACCAC 7 CGGAAAGGAAGACCAC 8 CGGCAAGGAGGACCAC 9 CGGCAAGGAGGACCAC 10 CAGGAAGGAGGACCAC 11 CAGCAAGGAGGACCAC 12 CAGCAAGGAGGACCAC 13 CAGGAAGGAGGACCAC 14 CAGGAAGGAGGACCAC 15 CGGCAAGGAGGACCAC 16 CAGGAAGGAGGACCAC 17 CAGGAAGGAGGACCAC 18 CGGCAAGGAGGACCAC 19 CAGGAAGGAGAACCAC 20 CAGGAAGGAGGACCAC 21 CAGGAAGGAGGACCAC 22 CAGGAAGGAGGACCAC TGGCAAGGAAGAC…

25. Amplification of pMCT118 pMCT118 Chromosome 1 PCR Or PCR Or any of 34 other length polymorphisms

26. Expected Results • Most people should show two fragments as bands following electrophoresis of PCR products • This is because each person has two copies of chromosome 1, one from each parent • As there are about 36 possible variations in length to the PCR product there should be about 362 or 1,296 different possible fragment patterns on gels

27. Previous Results

28. More Previous Results 10,000 Size (bp) 1,000 638 bp 530 bp 57 mm 1,857 bp 81 mm 87 mm 1,058 bp 929 bp 100 50 70 90 110 130 Distance Traveled (mm) 128 mm 383 bp D= 1/log10 MW MW is directly proportional to length in DNA 102 mm 642 bp - 530 bp = 112 bp 111 mm 112 bp = 16 bp x 7 Thus, the large fragment has 7 pMCT118 repeats more than the short fragment

29. The End