PCR Flashcards
What is PCR
To amplify a small length of DNA into numerous identical copies
What is the purpose of PCR
give examples
Forensic Testing
Paternity testing
Detecting mutations
What is a primer
A primer is designed as a short single stranded DNA sequence that acts as a starting point for DNA synthesis
What do you put in the PCR machine?
.DNA sample
.Taq polymerase
.Nucleotide bases
.DNA primers
MgCl2( it is a cofactor for DNA polymerase)
Why is there a buffer to maintain the pH level?
Because as number of Deoxyribose nucleic acid increases the more the solution gets acidic, which then the Taq polymerase might not be working best in different pH levels.
What is the relative tempreature for Denaturation step?
90-95c
What happens in the Denaturation step
The DNA is heated and breaks the hydrogen bonds, making single stranded molecules.
What is the relative temp for Annealing step
50-55c
What happens in the anealling step
The DNA strands are cooled allowing primers to bind to the complemantry sequences in the DNA.
What relative tempreture is used for the Elongation step?
72C
What are the exact steps for PCR?
- Denaturation, DNA is heated to 90c-95c to break the hydrogen bonds between the double strands to make them single stranded.
- Annealing The single stranded DNA is cooled down to 50c-55c to allow the primers to bind to its complementary sequences.
- Elongation Dna is reheated to 72C, for the enzyme Taq polymerase to work in optimal conditions. TAQ polymerae binds to the Primers and starts to make a new complementary strand of DNA.
- Repeat Repeat the PCR cycle repeatdly.
What is formula for the number of DNA (DOUBLE STRANDED) is created per PCR cycle?
x= 2^n
two to the power of the pcr cycle number
Why does the phosphate backbone not break during the first step of PCR which is?
Denaturation step breaks H bonds because it is weaker than the covalent bonds on the phosphate backbone.
Primers are often designed specifically for annealing by synthesising it. TRUE OR FALSE?
true
What are the two primers needed for the coding strand and template strand?
Foward primer for template strand and Reverse Primer for coding strand
What does the foward primer do
It binds to the start codon at the 3’ end of the template strand. TAQ polymerse then makes the strand the same direction RNA polymerase creates mRNA
What happens to enzymes if the tempreture was raised and decereased
What to mention in the answrr
Talk about the relation to rate of reaction
The enzyme denatures only past optimal
Talk about confirmational shape change in active site
Talk about if lowered temp, there wont be enough kinectic energy
Why do you need two different primers in PCR
You need two different because the complementary sequences are differnet at 5’ end of the coding and template strand.
When do you use PCR for and when you do use gene cloning for
PCR is used when small samples of DNA is available for labortaotry testing/forensics whereas Gene cloning is used to make copies of larger sections of DNA, whcich is used to insert DNA into a cell and produce a protien product in large quantaties.
NOTE: PCR is much quicker compared to gene cloning.
