21.3 In vitro cloning, polymerase chain reaction Flashcards
What is the polymerase chain reaction
Method of copying fragments of DNA
Process is automated so is rapid and efficient
What does polymerase chain reaction require to be present
- DNA fragment needs to be copied
- DNA polymerase needs to be present
- Primers
- Nucleotides
- Thermocycler
What does DNA polymerase do in the chain reaction and what is it
What do primers do and what are they
- It is an enzyme that can join together thousands of nucleotides
Primers are short sequences of nucleotides that have a set of bases complimentary to those at one end of each of the two DNA fragments
What is a thermocycler
A computer controlled machine that varies temperature precisely over a period of time
What is the first stage of polymerase chain reaction
DNA strand is separated:
The DNA fragments, primers and DNA polymerase are placed in a thermocycler
Temperature is increased to 95 degrees causing the two strands of DNA to separate as hydrogen bonds break
What is stage 2 of polymerase chain reaction, once DNA strands have separated
Mixture is cooled to 55 degrees causing primers to join to their complimentary bases at the end of DNA fragment
The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain
Stage 3 of polymerase chain reaction, once primers have given starting point for DNA polymerase
Temperature increased to 72 degrees which is optimum temperature for DNA polymerase
DNA polymerase adds complimentary nucleotides along each of the separated DNA strands
It begins at primer of both strands and continues till end of the chain
How many copies of the original fragment does polymerase chain reaction give
It gives two initially because both strands are copied simultaneously
Then once these are complete, process repeats by putting them through temperature cycle again to make 4 strands
So we can do this many times to produce millions of copies of DNA
In vitro cloning is rapid, why is this an advantage over in vivo
If only a tiny amount of DNA is available it can be quickly increased by this reaction.
This is good in forensic analysis of crime scene evidence as no time is wasted
However in vivo cloning will take weeks to do
What is another advantage of in vitro cell cloning over in vivo
In vitro doesn’t require living cells
All that is required is a base sequence of DNA that needs amplification
What are advantages of in vivo cell cloning over in vitro
Contamination
Contamination can occur in in vitro because it clones all DNA.
However in vivo does not have contamination because a gene that has been cut by the same restriction endonuclease can match the sticky ends of cut open plasmid.
What are advantages of in vivo cell cloning over in vitro
Accuracy
In vivo has very few errors compared to in vitro which can copy some DNA inaccurately
What are advantages of in vivo cell cloning over in vitro
When introducing gene into an organism
In vivo involves the use of vectors as once we introduce gene to plasmid it can be used to deliver gene into another organism eg another human
In vitro can’t
What are two other advantages of in vivo cell cloning over in vitro
- Produces transformed bacteria that can manufacture proteins for commercial or medical use
- Cuts out specific genes so very precise procedure
