Polymerase Chain Reaction
Introduction
Types
Applications
Polymerase Chain Reaction
Artificial amplification of a DNA sequence by repeated cycles of replication and strand separation
A process of DNA cloning
Requirements
1) DNA template
o DNA sequence to be amplified
o Some prior knowledge of the nucleotide sequences in the regions flanking the target sequence is required to make DNA primers
2)Amplimers
o Oligonucleotide primers
o DNA sequence
o 18-20 bp
o Hybridize with complementary DNA sequences both upstream and downstream of the desired sequence region
o Act as starting points for synthesis of new DNA strands
3)Polymerase enzyme
o DNA polymerase
o Heat stable
4) Resistant to temperatures at which denaturation takes place
o Derived from thermophilic bacteria Thermus aquaticus
5) Taq polymerase
o Catalyze polymerization of new DNA strands
6) dNTPs
o Deoxyribose nucleoside triphosphate (deoxynucleotides)
o Substrate for making new DNA strands
7) PCR Machine
o Thermocyclers
o Cycles around several different temperatures
Steps
1. A target sequence is chosen on the DNA. The sequence of the ends of the DNA should be known.
2. Two DNA primers, complementary to the 3' ends of the target DNA, are synthesized for each strand.
3. The DNA molecule carrying the target sequence is denatured by heat at 90-95 C for 20 seconds.
4. A reaction mixture containing all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) and a thermostable DNA polymerase is added.
5. The mixture is allowed to cool to a lower temperature (50-65 C). Each strand of DNA molecule becomes annealed with an oligonucleotide primer complementary to either end of the target sequence. Primer annealing takes 20 seconds.
6. The temperature is raised to 60-75 C and primers are extended by the action of DNA polymerase for 30 seconds. The polymerase synthesizes complementary sequence the 5' to 3' direction away from each of the primers.
7. The mixture is heated again at 90-95 C to denature the molecules and separate the strands and the cycle repeated. Each new strand then acts as a template for the next cycle of synthesis.
A typical thermal cycle might be as follows:
1. Heat denaturation at 94 C for 20 seconds
2. Primer annealing at 55 C for 20 seconds
3. Primer extension at 72 C for 30 seconds
Applications of PCR
1. Amplification of small amounts of DNA for further analysis by DNA fingerprinting.
2. Analysis of ancient DNA from fossils.
3. Mapping the human (and other species) genome.
4. Isolation of a particular gene of interest from a tissue sample.
5. Generation of probes: large amount of probes can be synthesized by this technique.
6. Production of DNA for sequencing: Target DNA in clone is amplified using appropriate primers and then its sequence determined. Helpful in conditions where amount of DNA is small.
7. Analysis of mutations: Deletions and insertions in a gene can be detected by differences in size of amplified
product
8. Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis, PCR is used to amplify
DNA from fetal cells obtained from amniotic fluid. PCR has also proved very important in carrier testing.
9. Detection of microorganisms: Especially of organisms and viruses that are difficult to culture or take long
time to culture or dangerous to culture.
10. Crucial forensic evidence may often be present in very small quantities, e.g. one human hair, body fluid
stain (blood, saliva, semen). PCR can generate sufficient DNA from a single cell.
Types:
Reverse transcription PCR - RNA strand is reverse transcribed using reverse transcriptase and the resulting cDNA is
amplified.
Real time PCR - Quantification of DNA occurs at the same time as amplification proceeds
Introduction
Types
Applications
Polymerase Chain Reaction
Artificial amplification of a DNA sequence by repeated cycles of replication and strand separation
A process of DNA cloning
Requirements
1) DNA template
o DNA sequence to be amplified
o Some prior knowledge of the nucleotide sequences in the regions flanking the target sequence is required to make DNA primers
2)Amplimers
o Oligonucleotide primers
o DNA sequence
o 18-20 bp
o Hybridize with complementary DNA sequences both upstream and downstream of the desired sequence region
o Act as starting points for synthesis of new DNA strands
3)Polymerase enzyme
o DNA polymerase
o Heat stable
4) Resistant to temperatures at which denaturation takes place
o Derived from thermophilic bacteria Thermus aquaticus
5) Taq polymerase
o Catalyze polymerization of new DNA strands
6) dNTPs
o Deoxyribose nucleoside triphosphate (deoxynucleotides)
o Substrate for making new DNA strands
7) PCR Machine
o Thermocyclers
o Cycles around several different temperatures
Steps
1. A target sequence is chosen on the DNA. The sequence of the ends of the DNA should be known.
2. Two DNA primers, complementary to the 3' ends of the target DNA, are synthesized for each strand.
3. The DNA molecule carrying the target sequence is denatured by heat at 90-95 C for 20 seconds.
4. A reaction mixture containing all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) and a thermostable DNA polymerase is added.
5. The mixture is allowed to cool to a lower temperature (50-65 C). Each strand of DNA molecule becomes annealed with an oligonucleotide primer complementary to either end of the target sequence. Primer annealing takes 20 seconds.
6. The temperature is raised to 60-75 C and primers are extended by the action of DNA polymerase for 30 seconds. The polymerase synthesizes complementary sequence the 5' to 3' direction away from each of the primers.
7. The mixture is heated again at 90-95 C to denature the molecules and separate the strands and the cycle repeated. Each new strand then acts as a template for the next cycle of synthesis.
A typical thermal cycle might be as follows:
1. Heat denaturation at 94 C for 20 seconds
2. Primer annealing at 55 C for 20 seconds
3. Primer extension at 72 C for 30 seconds
Applications of PCR
1. Amplification of small amounts of DNA for further analysis by DNA fingerprinting.
2. Analysis of ancient DNA from fossils.
3. Mapping the human (and other species) genome.
4. Isolation of a particular gene of interest from a tissue sample.
5. Generation of probes: large amount of probes can be synthesized by this technique.
6. Production of DNA for sequencing: Target DNA in clone is amplified using appropriate primers and then its sequence determined. Helpful in conditions where amount of DNA is small.
7. Analysis of mutations: Deletions and insertions in a gene can be detected by differences in size of amplified
product
8. Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis, PCR is used to amplify
DNA from fetal cells obtained from amniotic fluid. PCR has also proved very important in carrier testing.
9. Detection of microorganisms: Especially of organisms and viruses that are difficult to culture or take long
time to culture or dangerous to culture.
10. Crucial forensic evidence may often be present in very small quantities, e.g. one human hair, body fluid
stain (blood, saliva, semen). PCR can generate sufficient DNA from a single cell.
Types:
Reverse transcription PCR - RNA strand is reverse transcribed using reverse transcriptase and the resulting cDNA is
amplified.
Real time PCR - Quantification of DNA occurs at the same time as amplification proceeds
