21.3 In vitro gene cloning - the polymerase chain reaction Flashcards
Why is the PCR rapid and efficient
Because it is automated
What things does the process require
- The DNA fragment to be copied
- DNA polymerase
- Primers
- Nucleotides
- Thermocycler
What is DNA polymerase
An enzyme capable of joining together tens of thousands of nucleotides in a matter of minutes
Where is the DNA polymerase found
From bacteria in hot springs therefore it is thermostable and able to withstand high temperatures
What are DNA primers
Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments
What are nucleotides
Contain each of the four bases found in DNA
What is a thermocycler
A computer controlled machine that varies temperature precisely over time
What are the 3 stages of the PCR
- Separation of the DNA strand - 95 degrees
- Addition (annealing) of primers - 55 degrees
- Synthesis of DNA - 72 degrees
Process of separating the DNA strand (1)
The DNA primers, fragments and DNA polymerase are placed in a vessel in a thermocycler. Temperature is increased to 95 degrees, causing the two strands of the DNA fragments to separate due to the breaking of the hydrogen bonds
Process of primer addition (2)
The mixture is cooled to 55 degrees, causing the primers to join to their complementary bases at the end of the DNA fragment.
The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides at the end of an existing chain. Primers also prevent the two strands from rejoining
Process of DNA synthesis (3)
The temperature is increased to 72 degrees. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain`
How many strands are prodcued
After the first cycle there are now two copies of the original fragment. Once the two DNA strands are completed, the process is repeated and this results in four strands.
Advantages of In Vitro gene cloning
- Extremely rapid
- Does not require living cells. Only a base sequence of DNA
Advantages of In Vivo gene cloning
- Useful where we wish to introduce a gene into another organism because of vectors
- It involves no risk of contamination, due to restriction endonuclease use
- Very accurate, PCR was 20% inaccurate at one point
- Cuts out specific genes
- Produces transformed bacteria that can be used to produce large quantities of gene products
- produces
What is the order of processes and temperature required for each
- Separation of the DNA strand - 95 degree
- Addition of primers - 55 degrees
- Synthesis of DNA - 72 degrees
