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
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
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
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
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
Polymerase Chain Reaction (PCR)
 dNTP mix
 Contains a mixture of dATP, dTTP, dCTP, dGTP
 Provides the nucleotides needed for replication
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
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
Polymerase Chain Reaction (PCR)
 The Reaction is broken down into 3 steps per cycle
 Denaturation
 Annealing
 Elongation
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
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.
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
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
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
Polymerase Chain Reaction (PCR)

Designing PCR primers

Sample sequence:
5’AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCAC
CATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAAC
GCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGC
GCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATG
CGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT
GTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTG
GCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTC
TACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA
ATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT
TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA
TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATG
TGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG
AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGA
TAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC
ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTA
GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG
GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC-3’


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
Polymerase Chain Reaction (PCR)
5’AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCAT
CATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCA
GCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCG
CGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGG
GTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGG
GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGA
TTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCAT
CAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTT
CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA
GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT
TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT
ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA
TGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCG
GTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA
TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT
CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGC
CTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGT
AGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA
ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’
Polymerase Chain Reaction (PCR)

5’-


Select forward primer using rules
AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCAC
CATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACG
CAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGT
GAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGG
CTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAG
CGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAG
CAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATC
CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC
CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC
AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT
TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG
CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG
AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT
AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAAC
GACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTT
CCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’
Write in 5’-3’ direction
Forward primer: 5’-GAGCTTGAAGGTAAGCCTATC-3’
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
5’-
AAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCA
TCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTC
AGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAG
CGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGG
GGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTG
GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGG
ATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCA
TCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTT
TCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA
GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT
TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT
ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA
TGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCG
GTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA
TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT
CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGC
CTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGT
AGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA
ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC -3’
 Potential Reverse primer sequence
 5’-CAGAATTTGCCTGGCGGCAGTAGC-3’
 3’-GTCTTAAACGGACCGCCGTCATCG-5’
 5’-GCTACTGCCGCCAGGCAAATTCTG-3’
 Reverse primer:
 5’-GCTACTGCCGCCAGGCAAATTCTG-3’