Topic 3 (techniques to amplify and separate DNA) Flashcards
(19 cards)
State a technique to amplify DNA and another one to separate DNA fragments based on length
PCR (Polymerase chain reaction) and Gel electrophoresis
What is PCR?
PCR is a plymerase chain reaction which artificially replicates DNA that produces multiple copies of it
What is gel electrophoresis?
A technique used to separate DNA molecules by length
Where is a polymerase chain reaction done?
in a thermocycler, which repeats cycles making replicas of DNA
How is the process of producing more DNA called?
DNA amplification
State all the components of PCR and function
Template: DNA that we want to amplify
Primers: attach to the DNA template
dNTP’s: nucleotides that will form the new DNA strand
Polymerase: synthesizes the new DNA strand (Taq DNA polymerase that resist heat)
Buffer: maintains the optimal pH and ions.
State the 3 stages of a whole cycle of PCR
Denaturation, annealing and elongation
Explain the first step of PCR
Denaturation: during 30 to 60 sec, DNA will stay at a temperature of 95ºC to break the hydrogen bonds (not covalent).
Explain the second step of PCR
Annealing: during 30 to 60 sec, the DNA will stay at a temperature of 54ºC to allow the primers to attach to the DNA strand.
Explain the 3 step of PCR
Elongation: during 30 to 60 sec, DNA will be at a temperature of 72ºC to let the Taq polymerase work, this polymerase adds 1000 nucleotides per minute.
Explain the results of PCR
In 1 hour it will make 30 cycles from 1 molecule of DNA to obtain 1 billion copies.
What is gel electrophoresis used for?
to separate DNA by length
State the components of gel electrophoresis
- A Gel with pores
- Holes near one end of the gel (wells)
- Electrodes at both ends (creates electric current)
- Electrolyte solution (allows passage of electricity)
Explain how gel electrophoresis function
- A solution that contains DNA is placed in the wells of the gel.
- An electric current is applied, the DNA moves through the gel to the positive charge (DNA is negative because of phosphate group)
- The smaller fragments will move more because they can easily avoid pores, the bigger fragments will not because the cannot avoid the pores.
- Various DNA molecules with different lengths are put on the marker well to compare with the samples.
- The gel is treated with a dye to make the DNA visible
- The samples are compared with the marker to estimate a length.
Explain the results of gel electrophoresis
DNA molecules of the same length will have moved the same, so they will form a visible band in lane. With the number of bands in a lane we can know how many different lengths of DNA were in the sample. Markers indicate the mixture of DNA fragments of known length which is used to estimate the length of other fragments.
Explain how PCR for coronavirus is used.
A swab is taken from the patient, which is put on a saline solution, in which is converted to DNA by reverse transcriptase. Then, amplification is done, specific primers that only attach to Covid are used, if the patient is negative they will not attach. If they attach and DNA is produced fluorescent markers attach to the DNA and indicate that the test is positive.
Explian the advantages and disadvantages of the testing used for coronavirus
Advantages: it is very sensitive, one molecule of RNA I turned into millions of DNA, therefore with a tiny quantity a sample can be tested. It is very specific, primers are specifically designed to attach to the viral DNA.
Disadvantages: it is a very extensive process, and it takes 4-6 hours to get a result.
What is Gel electrophoresis for DNA profiling in paternity tests
STR’s, short tandem repeats, a technique to know whose parent is a child by examining DNA sequences. Ex: the sequence AGAT repeats between 5 and 20 times.
Explain Gel electrophoresis for DNA profiling in paternity tests
STR’s: the sample is obtained, the DNA is isolated from the sample. Then it is amplified by PCR, after it is separated by gel electrophoresis depending on the amount of repetitions. Then DNA profiles of the child, mother and father are compared. So, if any bands in the child’s profile does not occur in the profile of either mother or father, another person must be the parent.
