1. ©2001 Timothy G. Standish Romans 5:17 17For 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. ©2001 Timothy G. Standish Polymerase Chain Reaction Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish 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. ©2001 Timothy G. Standish 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. ©2001 Timothy G. Standish 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. ©2001 Timothy G. Standish Initiation - Forming the Replication Eye 3’5’ 3’5’ 3’ Origin of Replication 5’ 3’ 5’ 3’ 5’ 3’

7. ©2001 Timothy G. Standish Leading Strand Lagging Strand 3’ 5’ 3’ 5’ Extension - The Replication Fork 5’ 3’ 5’ 3’ 5’ Single-strand binding proteins DNA Polymerase Okazaki fragment RNA Primers Primase 5’ 3’ 5’ Helicase

8. ©2001 Timothy G. Standish Functions And Their Associated Enzymes è Ligase Joining nicks è DNA Polymerase Polymerizing DNA è Primase Providing primer EnzymeFunction è Helicase è SSB Proteins è Topisomerase Melting DNA

9. ©2001 Timothy G. Standish 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. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 5’3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’3’ 5’

11. ©2001 Timothy G. StandishPCR Melting 94 o C Temperature 100 0 50 T i m e 5’3’ 5’

12. ©2001 Timothy G. StandishPCR Melting 94 o C Temperature 100 0 50 T i m e 3’5’ 3’ Heat

13. ©2001 Timothy G. StandishPCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 3’5’ 3’ 5’ Melting 94 o C

14. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ Heat 5’

15. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

16. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’ Heat

17. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

18. ©2001 Timothy G. Standish Fragments of defined lengthPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

19. ©2001 Timothy G. Standish DNA Between The Primers Doubles With Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64

20. ©2001 Timothy G. Standish More Cycles = More DNA Number of cycles 0 10 15 20 25 30 Size Marker

21. ©2001 Timothy G. Standish Theoretical Yield Of PCR Theoretical yield = 2 n x y Where y = the starting number of copies and n = the number of thermal cycles = 107,374,182,400 If you start with 100 copies, how many copies are made in 30 cycles? 2 n x y = 2 30 x 100 = 1,073,741,824 x 100

22. ©2001 Timothy G. Standish How The Functions Of Replication Are Achieved During PCR è N/A as fragments are short Joining nicks è Taq DNA Polymerase Polymerizing DNA è Primers are added to the reaction mix Providing primer PCRFunction è Heat Melting DNA

23. ©2001 Timothy G. Standish 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. ©2001 Timothy G. StandishpMCT118 12 CAGCAAGGAGGACCAC 13 CAGGAAGGAGGACCAC 14 CAGGAAGGAGGACCAC 15 CGGCAAGGAGGACCAC 16 CAGGAAGGAGGACCAC 17 CAGGAAGGAGGACCAC 18 CGGCAAGGAGGACCAC 19 CAGGAAGGAGAACCAC 20 CAGGAAGGAGGACCAC 21 CAGGAAGGAGGACCAC 22 CAGGAAGGAGGACCAC TGGCAAGGAAGAC… …GCTCAGTGTCAGCCCA 1 AGG-AAGACAGACCAC 2 AGGCAAGGAGGACCAC 3 CGGAAAGGAAGACCAC 4 CGGAAAGGAAGACCAC 5 CGGAAAGGAAGACCAC 6 AGGCAAGGAGGACCAC 7 CGGAAAGGAAGACCAC 8 CGGCAAGGAGGACCAC 9 CGGCAAGGAGGACCAC 10 CAGGAAGGAGGACCAC 11 CAGCAAGGAGGACCAC

25. ©2001 Timothy G. Standish Amplification of pMCT118 Chromosome 1 pMCT118 PCR Or Or any of 34 other length polymorphisms

26. ©2001 Timothy G. Standish 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 36 2 or 1,296 different possible fragment patterns on gels

27. ©2001 Timothy G. Standish Previous Results

28. ©2001 Timothy G. Standish 10,000 90 100 5011013070 1,000 Size (bp) Distance Traveled (mm) More Previous Results 1,058 bp 929 bp 383 bp 1,857 bp 81 mm 87 mm 128 mm 57 mm 638 bp 530 bp 111 mm 102 mm Thus, the large fragment has 7 pMCT118 repeats more than the short fragment 642 bp - 530 bp = 112 bp 112 bp = 16 bp x 7 MW is directly proportional to length in DNA D= 1/log 10 MW

29. ©2001 Timothy G. Standish