Polymerase Chain Reaction Flashcards
What is PCR?
PCR allows tiny fragments of DNA to be amplified vitro by repeated cycling of heating and cooling.
Specific target sequences of DNA are replicated out with the body of the organism, in a laboratory.
Amplification
Small samples of DNA are copied enough so that they can be more easily analysed..
How much does one cycle of PCR increase number of DNA molecules?
They double per cycle.
Requirements for PCR
Primers, Supply of DNA nucleotides, DNA sample, heat tolerant DNA polymerase, buffer solution to maintain optimum pH is also added to the PCR tube.
A thermal cycles is then used to run the process.
Primers for PCR
As with replication inside the cell, DNA amplification by the PCR requires a primer to allow DNA polymerase to start synthesis of the new strand.
Sequence specific primers are designed by the experimenter to match the beginning and end of the target DNA fragment. The primer can then be manufactured by a machine.
How does the primer only target the specific sequence?
Designed with sequences which are complementary to DNA opposite strands of the template just at the ends of the region to be copied.
Heat tolerant DNA polymerase
The DNA polymerase used in PCR most often is Taq polymerase. The enzyme was first isolated from the bacteria Thermophilus aquiticus, which live on the edge of hydrothermal vents and is therefore adapted to high temperatures.
Why is heat tolerant DNA polymerase required?
Required since PCR relies on multiple cycles of replication in which heat is used to separate DNA strands. These high temperatures would typically denature typical enzymes.
PCR Process - Cycle 1, Stage 1
All requirements are added to PCR tube.
This is heated to between 92-98 degrees Celsius to denature the DNA and separate the strands of sample DNA by breaking hydrogen bonds.
PCR Process - Cycle 1, stage 2
PCR tube is cooled to between 50 and 65 degrees Celsius to allow primers to bind to target sequences.
PCR Process - Cycle 1, stage 3
The temperature is the raised to between 70 and 80 degrees Celsius for heat tolerant DNA polymerase to add free nucleotides to the 3’ end of the newly forming strand, starting at the primer, using complementary base pair rules.
Cycle 2
Entire process repeats itself.
Temperatures for PCR
92-98 degrees Celsius to allow the two strands to separate.
50-65 degrees Celsius to allow primers to bind to target sequences.
70-80:degrees Celsius for heat tolerant DNA polymerase replicates the target region of DNA.
Uses of PCR No. 1
Forensic science
A tiny quantity of genetic material found at a crime scene can be amplified to provide enough material for various tests to identify suspects and solve crimes.
Uses of PCR No. 2
Disease detection
DNA sequences that are known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of pre-natal diagnosis in embryonic cells of these disorders.
