DNA manipulation techniques Flashcards
(31 cards)
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.
The creation of a recombinant DNA molecule requires several steps that involve the use of enzymes.
a. What role is played by a restriction enzyme in this creation?
b. What role is played by the enzyme DNA ligase?
a. A restriction enzyme that (1) cuts double-stranded DNA to produce overhanging ‘sticky’ ends and (2) has a recognition site present in an appropriate location in the DNA from both sources is selected. Sticky ends are complementary so, when DNA fragments from the two sources are mixed, they can pair.
b. DNA ligase is required to permanently join the paired fragments of recombinant DNA. (Ligase catalyses the formation of strong covalent bonds of the sugar–phosphate backbones of the DNA.)
Define:
a) a primer.
b) DNA ligase.
c) DNA polymerase.
d) gel electrophoresis.
a) Primers are short single-stranded DNA or RNA molecules which are complementary to a sequence of nucleotides on a particular DNA molecule.
b) DNA ligase is an enzyme that joins the phosphate group and sugar group in the backbone of the DNA molecule. To join a piece of DNA to a molecule of DNA, a DNA ligase is required.
c) DNA polymerase is an enzyme involved in DNA replication by controlling the addition of complementary nucleotides.
d) Gel electrophoresis is a technique used to separate out molecules of DNA (or other charged molecules) according to size.
An example of recombinant DNA molecules includes DNA molecules formed…?
By splicing DNA from one species into that of a second species.
Give a conclusion of electrophoresis which could be drawn about the sample taken from the crime scene.
Two suspects matched the sample taken from the crime scene.
- the sample is not from the victim
- the sample could be from either suspect.
NOT: - both suspects must be guilty
- both suspects are identical twins
- the victim wasn’t at the crime scene
What further action would you recommend to the forensic scientists to confirm the results of electrophoresis which suggest an individual is responsible for a crime.
- apply the same process to a different gene locus
- use another suitable DNA technique, such as DNA sequencing
- use another forensic method, such as blood analysis or fingerprinting.
What does DNA manipulation refer to?
Altering an organism’s DNA by either adding new DNA or editing existing DNA
What is a restriction enzyme?
- Endonuclease is a tool used by genetic engineers to cut DNA
- cuts DNA at precise sequences of 4-8 base pairs called recognition sequences. Once recognised the enzyme binds to DNA and cuts it in a fixed and predictable way
Where do restriction enzymes come from?
Occur naturally in Bacteria, thought to have evolved as a defence mechanism against viruses.
What is a recognition sequence?
The position where a cutting enzyme can snip its recognition sequence and is where a particular order of nucleotides occurs
