Week 3 Flashcards
What is the purpose of PCR? (polymerase chain reaction)
- to target and amplify the specific gene region of interest so it can be read/sequenced on a sequencing machine.
What are the 5 main ingredients to PCR?
- primers
- thermostable DNA polymerase enzyme (taq pol)
- genomic DNA (template) - things we want it to replicate (DNA extraction)
- some co-factor chemicals (mg2+, buffer, KCl) - help other things do their job
- deoxyribose nucleoside triphosphates (dNTPs)
What are dNTPs? What are the types?
- They are similar to a nucleotide, but has 3 phosphate groups instead of one (in a nucleotide)
- dATP, dGTP, dTTP, dCTP
What is a nucleotide? Nucleoside? dNTP?
What do dNTPs do?
- They build new strands of DNA (by DNA polymerase)
- need all types of them in PCR
How does DNA replication work by DNA polymerase?
- dna polymerase will bind growing chain and dNTPs in solution by a phosphodiester bond, and extra phosphates fall off into solution.
What are PCR primers? What determines PCR product size?
- single-stranded DNA fragments complementary to the sequences flanking the target region of amplification. The distance between the primer binding sites determines the size of the PCR product.
How do PCR primers work (walk through)?
- template becomes single stranded and primers bind on each strand.
- enzyme binds on after primers (taq polymemrase) by incorporating dNTPS to create a growing strand.
Which direction does taq polymerase extend?
- 5’ to 3’ direction
Three steps of PCR?
- denaturation of target at 95C
- annealing of primers at 50-56C (primers bind to each strand)
- ## extension (synthesis) of new DNA at 72C
Is annealing temperature variable or not in PCR?
- variable depending on G+C content
What is a thermocycler also called? What does it go through?
- A PCR machine.
- denaturing, annealing, extension.
What does a thermocycle profile look like?
What happens by the third cycle of PCR? Why?
- get double-stranded DNA of the desired length of our target sentence
- because primer keeps replicating until it falls off the molecule in the first steps.
- Then the molecules that you find will only be the ones that are cut at one end, so then the primer might bind to one that has been cut at one end again, then it won’t replicate further than that cut end obviously, giving you your perfect size.
Considerations for the primers in PCR?
- primer pairs must have similar annealing temperatures
- the primer sequence must be unique to that region.
- primers should not anneal to themselves or other primers in the mix. (we go with what’s going to bind with the DNA of interest most of the time.
Where should primers attach?
- conserved regions (where the region is the same across tested individuals)
- variable regions (polymorphisms (differences) areas) are regions we want to replicate, but we need to start at the conserved regions.
What are specific primers useful for?
- one species
- useful for detection (presence/absence, even without sequencing using gel electrophoresis.
- e.g. detection of diseases, species confirmation.
What happens if you get a band with a specific primer in gel electrophoresis after PCR?
- that you have amplified DNA from that species. If there is no band, not present.
What are universal primers?
- amplify same target DNA in different species (can be broad (all frogs), or more specific (within a genus). Depending on design
What is degeneracy in universal primers?
- contain degeneracy, which make them compatible with a variety of target sequences.
- ## if several different ones of the conserved regions, then we have different symbols, to give us a mixture of primers to bind to all species selected (based on slightly different conserved regions)
What do we use in gel electrophoresis?
- 1% agarose powder dissolved in TBE buffer (stable buffer) which creates a gel. We put sort of teeth in, which creates wells, which we load the samples into.
- DNA is also mixed with dye in the wells at the negative terminal
What is the point of gel electrophoresis?
- we have a ladder to tell the size of the fragment
- ## the current pulls the DNA across and smaller sized fragments will travel further.
What do we want to see in PCR?
Things that can go wrong with PCR?
- you get no bands: no DNA (bad extraction), primers cant bind (so redesign them), annealing temperature too high (nothing could bind) so lower it, dodgy old reagents (polymerase or dNTPs) so replace. OR inhibitors are present (like in slimy creatures) so dilute the DNA - dilutes inhibitor.
- You get bands of the wrong size: primers are binding at multiple or different locations in the genome: if you can’t see your target band, redesign primers. If you can see it, increase annealing temp, redesign primers to make them more specific, or try touchdown PCR (higher annealing temp in first few cycles promotes specificity).
What is touchdown PCR?
- higher annealing temperature (Ta in first few cycles to promote specificity).
What is primer dimer?
Bright smeary bands occur below the ladder and these are primer dimer. Which is left over unused primers. Not a huge problem. PCR purification can get rid of these.
