Final Biot63 Flashcards
What are PCR steps?
1) denature (95 C)
2) annealing (54 C)
3) extension (72 C)
What is the most problematic step when trying to clone a new gene?
It is the annealing of primers, because if the primers stick to themselves or if the template changes and the sequence is no longer the same, the primers may be annealed to the incorrect position, amplifying the wrong area of the DNA template.
What are oligonucleotides?
Oligonucleotide primers are complimentary to the ends of target DNA you want to amplify.
What is a consensus sequence?
a sequence of nucleotides or amino acids similar or identical between regions of homology in different but related DNA, RNA, or protein sequence.
Why is it important to include a negative control when doing PCR?
The negative control has no template DNA, so nothing should amplify. If something does amplify, it is due to contamination in the reagents, and it indicates that the bands in the other samples are likely contaminated as well.
What problems might you encounter when using genomic DNA as a PCR template?
Shear size of genomic DNA means primers might bind to other sites.
What special property must be possessed by the DNA polymerase used in PCR, and why is this important for doing PCR?
It must be thermal stable so it doesn’t get denatured by the heat necessary to separate the DNA strands.
Explain the use of primers and why are two necessary.
Primers are used to determine the DNA sequence to be amplified. A primer is a strand of short nucleic acid sequences that serves as a starting point for DNA synthesis. DNA polymerases can only add new nucleotides to an existing strand of DNA. One primer can only allow for the synthesis of one strand, so you need two.
What relationship must exist between the two primers used in PCR?
They must be designed to match each sequence of the target sequence.
Explain the overall process of PCR.
1) denature (95 C) - DNA is heated to 95 degrees Celsius. This breaks the weak hydrogen bonds that hold the DNA strands together, allowing the strands to separate creating a single stranded DNA.
2) annealing (54) - The mixture is cooled to anywhere between 45 - 72 degrees Celsius. This allows the primers to bind to their complementary sequence in the template DNA.
3) Extension (72) - The reaction is then heated to 72 degrees Celsius, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.
How does a band of DNA arises on electrophoresis after PCR?
The band of DNA arises when protein is added to the tracking dye and they migrate to the positive charge of the electrophoresis current. Since DNA has a very high MW, it migrates very slowly, therefore appearing at the very top of the gel as thick, and bright band.
How do you visualize DNA bands in an agarose gel?
By adding the loading buffer.
Why is it not necessary to add new DNA polymerase and primers for each new cycle of PCR?
Because DNA polymerase is an enzyme and as such it remains chemically unchanged at the end of the reaction.
Why was discovery of Taq DNA important for the development of PCR?
Because Taq is able to withstand high temperatures so it doesn’t let DNA denature during PCR procedure.
