Technology used for identifying genetic mutations Flashcards
Name the different methods used to identify genetic mutations
Polymerase chain reaction
Gel Electrophoresis
Amplification refractory mutation system (ARMS)
Restriction fragment length polymorphism (RFLP)
DNA sequencing
What does ARMS stand for?
Amplification refractory mutation system
What does RFLP mean?
Restriction fragment length polymorphism
Explain the process of polymerase chain reaction.
- Denature the DNA by heating to 93-95 degrees Celsius
- Anneal primers to the DNA- 50-70*c
- Extend- synthesis new DNA strand from the primers 70-75*c
repeat 20-30 times
Briefly explain how Gel electrophoresis works
DNA is -ve, so can be separated by size using an electrical current.
Apply the electrical current to the agarose gel that DNA fragments are on/in. The smallest fragments will move the furthest.
You can compare specimens to see which “fragments” (genes?) came from which parent.
What are the advantages of gel electrophoresis?
It is quick, easy to use, robust and sensitive
What are the disadvantages of gel electrophoresis?
Sequence information is needed
limited amount of product
Outline how amplification refractory mutation system works
You use mutant and normal primers with single strands of DNA. If the mutant primer anneals to the sample, then the gene has mutated.
Gel electrophoresis is then required for analysis.
What are the advantages of ARMS?
Cheap
labelling is not required
What are the disadvantage of ARMS?
Primer design is critical to the process and electrophoresis is required.
Describe how Restriction fragment length polymorphism works.
Changes in the DNA sequence cause restriction enzymes to cut DNA at different point (or not cut at some points).
This results in different fragment lengths, which can then be analysed by gel electrophoresis.
What are the advantages of RFLP?
simple
cheap
non-radioactive
What are the disadvantages of RFLP?
Requires gel electrophoresis, not always feasible.
Explain a simplified method of Sanger DNA sequencing
Put single stranded DNA (multiples of the same fragment), DNA primers, DNA polymerase and the 4 types of DNA bases in 4 test tubes.
Then add a different dideoxynucleotide (A, G, C or T) to each test tube.
The dideoxynucleotides will cause termination of synthesis of a complementary strand of DNA when added to the strand.
This means different length fragments will be produced.
The put the fragments from each test tube in a different lane/line of gel.
Carry out gel electrophoresis
When the fragments have been spread out, you will be able to read off the original DNA sequence, as the first one will be the bases which has a fragment that travelled the furthest towards the positive electrode (as it will be the shortest fragment)
