Molecular Techniques in Medicine
The Polymerase Chain Reaction
PCR
§ PCR performs the chemistry of DNA duplication in vitro: outside of the cell.
§ Numerous PCR applications make this process a staple in most biology laboratories.
§ Understanding properties of DNA polymerases helps understanding PCR.
PCR was discovered by Kary Mulli On a long motorcycle drive Mentally visualized the
process
Nobel Prize in Chemistry 1993
The three main steps of PCR
Step 1: Denaturation - Denature DNA
At 95°C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Annealing - Primers Anneal
At 40°C- 65°C, the primers anneal (or bind to) their complementary sequences on the
single strands of DNA
Step 3: Extension - DNA polymerase Extends the DNA chain
At 72°C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends
of the primers.
Initially PCR used the Klenow fragment of E. coli DNA polymerase inactivated by
high temperatures.
Required a thermostable DNA polymerase - Taq (Ideal temperature 720 C)
DNA polymerase from Thermus aquaticusa thermophilic eubacterial microorganism
isolated from a hot spring in Yellowstone National Park
§ In vitro amplification of a targeted in a DNA strand/genome.
Mimics cellular replication.
§ A heat stable DNA polymerase is required.
Use an equipment called thermal cycler.
§ Needs primers - a short DNA sequence, which is complementary to the ends of
the target region to be amplified.
, Molecular Techniques in Medicine
Thermal cycling
• A PCR machine controls temperature
Typical PCR goes through three steps
• Denaturation
• Annealing
• Extension
Denaturation of DNA Melt DNA duplex. 90-95 0C for 30-60 sec.
This occurs at 95 ºC mimicking the function of helicase in the cell.
Primer annealing - Annealing of primers to single stranded DNA. 50-650C
for 30-60 sec.
§ Two primers are supplied.
§ They bind to the complementary region.
§ As the DNA cools, they wedge between two template strands
§ Optimal temperature varies based on primer length etc.
Typical temperature from 40 to 60 0C
What is a Primer..?
• Short chains of bases that hybridize to a target piece of DNA.
• Primers determine the DNA fragment to be amplified in the PCR process.
Reverse Primer
Forward Primer
The Polymerase Chain Reaction
PCR
§ PCR performs the chemistry of DNA duplication in vitro: outside of the cell.
§ Numerous PCR applications make this process a staple in most biology laboratories.
§ Understanding properties of DNA polymerases helps understanding PCR.
PCR was discovered by Kary Mulli On a long motorcycle drive Mentally visualized the
process
Nobel Prize in Chemistry 1993
The three main steps of PCR
Step 1: Denaturation - Denature DNA
At 95°C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Annealing - Primers Anneal
At 40°C- 65°C, the primers anneal (or bind to) their complementary sequences on the
single strands of DNA
Step 3: Extension - DNA polymerase Extends the DNA chain
At 72°C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends
of the primers.
Initially PCR used the Klenow fragment of E. coli DNA polymerase inactivated by
high temperatures.
Required a thermostable DNA polymerase - Taq (Ideal temperature 720 C)
DNA polymerase from Thermus aquaticusa thermophilic eubacterial microorganism
isolated from a hot spring in Yellowstone National Park
§ In vitro amplification of a targeted in a DNA strand/genome.
Mimics cellular replication.
§ A heat stable DNA polymerase is required.
Use an equipment called thermal cycler.
§ Needs primers - a short DNA sequence, which is complementary to the ends of
the target region to be amplified.
, Molecular Techniques in Medicine
Thermal cycling
• A PCR machine controls temperature
Typical PCR goes through three steps
• Denaturation
• Annealing
• Extension
Denaturation of DNA Melt DNA duplex. 90-95 0C for 30-60 sec.
This occurs at 95 ºC mimicking the function of helicase in the cell.
Primer annealing - Annealing of primers to single stranded DNA. 50-650C
for 30-60 sec.
§ Two primers are supplied.
§ They bind to the complementary region.
§ As the DNA cools, they wedge between two template strands
§ Optimal temperature varies based on primer length etc.
Typical temperature from 40 to 60 0C
What is a Primer..?
• Short chains of bases that hybridize to a target piece of DNA.
• Primers determine the DNA fragment to be amplified in the PCR process.
Reverse Primer
Forward Primer
