L13 PCR Flashcards
What is PCR?
- Amplify DNA in vitro (in a tube).
- Specific - will only get amplification of your selected sequence.
- Selective - can amplify a specific sequence from a mixture of DNA sequences
What features are needed when making a new strand? (4)
It must be:
1. Complementary nucleotide base pairing (A-T and G-C)
2. Double-stranded
3. Antiparallel
4. Semi-conservative (template and newly synthesised daughter)
What do we need for PCR replication? (4)
- Need to unwind DNA
- Need a primer
- Polymerase enzyme
- New nucleotides
What is the basic principle of PCR?
PCR is a cyclic process that amplifies a specific DNA sequence. Each cycle involves three steps:
- Denaturation: The double-stranded DNA is heated to separate the two strands.
- Annealing: Primers, short single-stranded DNA sequences complementary to the target sequence, bind to the template DNA.
- Extension: DNA polymerase extends the primers, synthesizing new DNA strands complementary to the templates.
How does PCR amplify DNA exponentially?
In each PCR cycle, the number of DNA molecules doubles. This exponential amplification is achieved by repeating the following steps:
* Denaturation
* Annealing
* Extension
After 30 cycles, you can theoretically generate over one billion copies of the target DNA sequence from a single starting molecule.
What do we need for PCR? (6)
- Template - desired DNA
- DNA polymerase - copies DNA
- Primers - DNA polymerase need free 3’-OH provided by a primer.
- dNTPs - DNA bases to make new strands
- Buffer - correct pH and ions
- Thermocycler - maintains appropriate temperature.
What is the challenge of using DNA polymerase in PCR?
The high temperatures required for denaturation (95°C) can inactivate most DNA polymerases. To overcome this challenge, thermostable DNA polymerases, such as Taq polymerase, are used in PCR. These enzymes are derived from thermophilic bacteria and can withstand high temperatures without denaturing.
What are the DNA polymerases used in PCR and why?
Taq polymerase is used because of its thermostability, extension rate( how fast it can replicate), processivity and fidelity.
Which polymerase is more effective than Taq and why?
Pfu with superior thermostability and higher fidelity.
What are the important features of PCR primers?
- Minimum size for specifity (17bp minimum)
- Specific to your template (primer is complementary)
- Come in pairs (Bind strand in opp orientation)
- Appropriate melting temperature (specificity)
How do primers work?
Primers come in pairs. One of each pair will bind top strand of DNA; one will bind bottom strand. In opposite orientations (3’ ends point towards each other)
What is primer Tm and how does it affect PCR?
- Tm: Temperature at which the primer will dissociate from the DNA template.
- Primer Tm: Usually design primers to have a Tm = 60-64°C.
- Primer Tm determines what annealing temperature (Ta) to use in your PCR cycle - should be about 5°C lower than 1 the Tm.
What happens if ta (annealing temperature) is right, too low or too high?
- Right - Primers bind to the specific sequence.
- Too Low - Primers may bind non specifically to other sequences
- Too High - Primers may not bind effeciently.
How do you calculate primer Tm?
Old-fashioned method (2+4):
Add 4°C for every G/C; add 2°C for every A/T
How can we ligate a PCR product directly into a vector?
1) PCR products have no 5’ phosphate:
* Make primers with phosphates
* Add a 5’ phosphate (T4 PNK)
* Rely on the 5’ phosphate in the vector
2) Taq adds a 3’ A overhang to its PCR products:
* Remove the 3’ overhang
* Use clever vectors
* Use a different enzyme
What are the advantages and disadvantages of incorporating restriction sites into primers?
- More effecient ligation:
* Sticky ends after restriction digest
* Vector can’t ligate to itself.
* No need for phosphatase treatment. - Directional - use different enzymes.
Explain the variant of PCR – (RT-PCR).
Overview:
1. RNA is reverse transcribed into DNA (copy / complementary DNA = cDNA)
2. PCR is used to amplify a specific cDNA sequence.
Potential uses:
* Molecular cloning of a protein coding cDNA sequence.
* Analysis of RNA sequence / expression.
What is the first step of RT-PCR: cDNA synthesis?
First strand synthesis:
1. Reverse transcriptase synthesis the first strand of cDNA.
2. Poly(dT) primers bind poly(A) tail of mRNA.
3. RNA is removed.
Explain second strand Synthesis?
- Synthesised by the Klenow fragment of DNA polymerase I
- The hairpin formed by RT acts as a primer
- The ssDNA loop can be digested by a nuclease
What is step 2 of RT-PCR?
PCR:
* Template = cDNA from step 1
* Just need the usual reagents: primers, dNTPs, DNA polymerase
* And a thermocycler
What is the other variant of PCR?
Quantitative PCR
How to measure qPCR product? (Step 1)
Fluorescent dye:
- SYBR Green
- Fluoresces when it binds dsDNA
- Fluorescence is proportional to amount of dsDNA
- Not sequence specific
How to measure qPCR product? (Step 2)
Fluorescent probes
- Sequence specific
- Can multiplex
- Fluoresces when displaced from template
What is the significance of Ct (or Cq) in qPCR?
- Cycle threshold = the point at which fluorescence exceeds background levels (before amplification levels)
- Fluorescence will increase during qPCR run (with more cycles)
- Difference between Ct values is a relative measure of which sample had most template to start off with
How do we calculate relative measure between Ct values and fold difference?
- We can use the difference in Ct values (ΔCt) between two samples to calculate relative amounts.
- For our example from before, ΔCt (A-B) = -5
- Fold difference = 2^(-ΔCt) = 2^5 = 32
(Therefore, A has 32x more template than B.)
