Polymerase Chain Reaction (PCR)

1. Polymerase Chain Reaction (PCR) • Developed in 1983 by Kary Mullis • Major breakthrough in Molecular Biology • Allows for the amplification of specific DNA fragments from genome or genetic sample of an organism • Benefit is that only a small amount of sample DNA needed

2. Polymerase Chain Reaction (PCR) • Reaction mimics DNA replication • Uses thermostable DNA polymerase (Taq Polymerase) • Every reaction contains some basic components • Template DNA • Primers • dNTP mix • Buffer • DNA polymerase

3. Polymerase Chain Reaction (PCR) • Template DNA • DNA which contains sequence to be amplified • Can be chromosomal, plasmid, linear, or circular • Only a small amount needed for reaction

4. Polymerase Chain Reaction (PCR) • Primers • Small oligonucleotides that provide the start points for DNA replication • One primer required for each strand in order to amplify double stranded DNA • Primers must be oriented facing each other in the 5’ to 3’ direction • Primers must be complementary to template sequence targeted for amplification

5. Polymerase Chain Reaction (PCR) • General rules for designing primers • Each primer should be 18-25 bases in length • Approximately 50% G/C content • Should contain a G or C at each end to anchor ends of primer • No runs of 3 or more of the same nucleotide • Ex. TAAAACG

6. Polymerase Chain Reaction (PCR) • dNTP mix • Contains a mixture of dATP, dTTP, dCTP, dGTP • Provides the nucleotides needed for replication

7. Polymerase Chain Reaction (PCR) • Buffer • Mimics physiological conditions for polymerase to function • There is variability of some buffer components which can alter success of the reaction • Mg2+ concentration required for function of polymerase • pH of final concentration • NaCl concentration • Optimize conditions for PCR using buffers that vary in these components

8. Polymerase Chain Reaction (PCR) • DNA polymerase • Purified bacterial enzyme that catalyzes DNA replication • Taq polymerase • Isolated from thermophilic bacteria • Can withstand high temperatures and repeated heating and cooling

9. Polymerase Chain Reaction (PCR) • The Reaction is broken down into 3 steps per cycle • Denaturation • Annealing • Elongation

10. Polymerase Chain Reaction (PCR) • Denaturation • Double stranded template DNA separated • Done by heating reaction to 95oC • Annealing • Primers bind to DNA template at a cooler temperature • Annealing temp determined by the primer sequence • Usually around 48-54°C

11. Polymerase Chain Reaction (PCR) • Elongation • Replication of targeted DNA sequence • Reaction is heated to 72oC to allow Taq polymerase to replicate DNA • Primers are essential to begin replication and the complementary strand is synthesized as an elongation of the primer • When replication occurs using genomic DNA as the template the complementary strand will be synthesized until the polymerase detaches from the template. Therefore early rounds of PCR yield products of variable sizes.

12. Polymerase Chain Reaction (PCR) • The cycle is repeated many times • The replicated DNA from previous cycles is used as a template for the next cycle of replication • The primers “bracket” the DNA target and after three rounds of replication target DNA fragments will begin to appear

13. Polymerase Chain Reaction (PCR) • A specialized piece of equipment known as a thermocycler is needed to run the reaction • Thermocycler • Computer controlled chamber that heats and cools the reaction rapidly • Ramping time- amount of time it takes thermocycler to go from one temperature to another • The faster the better

14. Polymerase Chain Reaction (PCR) • Typical Thermocycler program for PCR is as follows: • 95oC – 5 minutes - Initial denaturation • 95oC – 40 seconds • 54oC – 45 seconds – Annealing • 72oC – 45 seconds • Repeat 30 times • 72oC – 10 minutes to complete elongation of any unfinished strands • 4oC – storage until reaction can be removed

15. Polymerase Chain Reaction (PCR) • Designing PCR primers • Sample sequence: • 5’- AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGC • GCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC-3’

16. Polymerase Chain Reaction (PCR) • Create primers to amplify 200 bp sequence • First find sequence of interest to amplify • You can pick any sequence to amplify within the sequence

17. Polymerase Chain Reaction (PCR) 5’-AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’

18. Polymerase Chain Reaction (PCR) • Select forward primer using rules 5’-AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’ • Write in 5’-3’ direction • Forward primer: 5’-GAGCTTGAAGGTAAGCCTATC-3’

19. Polymerase Chain Reaction (PCR) • Select Reverse primer • Must use the reverse complement in order to get correct sequence for priming • Select sequence, write the complement of the sequence , reverse the complement of the sequence

20. 5’-AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’

21. Potential Reverse primer sequence • 5’-CAGAATTTGCCTGGCGGCAGTAGC-3’ • 3’-GTCTTAAACGGACCGCCGTCATCG-5’ • 5’-GCTACTGCCGCCAGGCAAATTCTG-3’ • Reverse primer: • 5’-GCTACTGCCGCCAGGCAAATTCTG-3’