DNA manipulation techniques Flashcards
Describe process of PCR. (4)
- heat DNA to approximately 90 °C or to separate strands
- cool to approximately 50 °C or to anneal/attach primers
- heat to approximately 72 °C or Taq/DNA polymerase copies strands
- repeat cycle.
What do the standards consist of, and what is their purpose?
The standards consist of fragments of known sizes their purpose is to enable estimation the size of the fragments in the samples.
- comparison of known to unknown.
Write a short paragraph to explain the phrase rational drug design.
- the analysis of a disease to determine a structure/aspect of the disease. A drug is then designed to mimic/block the action of the disease-causing agent
- a drug developed to act specifically on an infective agent/enzyme. This then binds and removes the capacity to cause disease
Why would a fragment cut by a restriction enzyme have blunt ends?
Because the cutting site * is in the middle of the recognition sequence, an equal number of bases (two) are on either side of the cut so the enzyme will make a blunt cut, 5’ AG*CT 3’.
In the complementary DNA strand, the reverse sequence 3’ TC*GA 5’ is cut at the directly opposite site/complementary base/same base pair and so the cut will look even.
What would make a rationally designed drug the most effective?
This drug has the most points which are complementary to the active site.
Explain why the DNA of each individual produces a different pattern of fragments after gel electrophoresis, even when the same restriction enzyme is used.
Different individuals having different DNA sequences so there may be a different number of cutting sites, resulting in different numbers or sizes of DNA fragments.
What does hybridisation mean?
The joining of complementary DNA from different sources
Explain why the DNA polymerase enzyme used is heat resistant.
- Since PCR is carried out at elevated temperature, the DNA polymerase enzyme that is used in the incubation mixture must be heat resistant so that it can be active.
- If it were not heat resistant, the high temperatures would inactivate the enzyme so that it could no longer catalyse its specific reaction. (The DNA polymerase enzyme that is used comes from a bacterial species that lives in hot springs.)
The polymerase chain reaction is often described as a series of ‘denature-bind-extend’ cycles. Why is it necessary to denature the double-stranded DNA fragment that is to be amplified?
- The DNA to be amplified is initially denatured so that it becomes single stranded.
- This is necessary to enable the later binding of primer molecules to each strand.
In this procedure, scientists select a particular restriction enzyme from an available range.
Explain the reason for their choice.
Explanation should refer to each enzyme having its own recognition sequence so the scientists need to choose one that cuts the DNA at the site they are interested in.
Explain why the same restriction enzyme must be used to cut the fragments.
- to produce the same sequences at the (sticky/asymmetrical) cut ends of the fragments from the different sources
- that the (sticky) ends from the differently-sourced fragments will join together by complementary base-pairing
- If different restriction enzymes were used, only the cut end from the DNA fragments from the same source would be able to rejoin by complementary base-pairing.
A DNA polymerase enzyme is involved in the Polymerase Chain Reaction (PCR) process. Explain the role of the polymerase enzyme in PCR.
- The enzyme DNA polymerase makes multiple copies of DNA OR amplifies the DNA.
- The enzyme replicates DNA by complementary base pairing OR by using the DNA as a template.
Each restriction enzyme cuts DNA at particular sequence of bases called the…?
Each restriction enzyme cuts DNA at particular sequence of bases called the recognition sequence.
Suggest why the use of restriction enzymes and gel electrophoresis is not possible with the HERDA mutation.
- there may not be a restriction enzyme available that cuts at the faulty position,
- the two strands are the same length and therefore can’t be separated using gel electrophoresis.
The difference in the gene is a one base change. The difficulty would be finding a suitable restriction enzyme that may cut the DNA to show this difference.
In PCR:
a. What occurs during the ‘bind’ phase?
b. What occurs during the ‘extend’ phase?
a. In the ‘bind’ phase of PCR, primers bind to opposite ends of the DNA strands to be amplified. Primers can do this because they are short single-stranded DNA sequences that are complementary to the opposite ends of these strands.
b. In the ‘extend’ phase of PCR, the DNA polymerase enzyme uses the primers as starting points and extends them to create new copies of double-stranded DNA.
