PCR

PCR

1. Ingredients:

   • DNA Template
     • Complementary DNA
       • Synthesized in the lab from RNA
     • Genomic DNA
       • Naturally Occuring
   • Left and Right Primers
     • short pieces of DNA made in the lab - custom built therefore can have any sequence you like
       • aka. designed by you and synthesised by supplier.
     • ~18-24 nucleotides long synthetic oligonucleotides
       • Through complementary base paring = one attaches to the top and the other to the bottom of your segment of interest
     • Necessary because = DNA can't just attach anywhere and start copying
     • area added in excess to the reaction
   • DNA Taq Polymeraze
     • synthesises new DNA strand complementary to the template using the excess nucleotides.
     • Taq - THERMOSTABLE and originally isolated from 'thermopiles aquaticus'
       • therefore can survive repeated high temperatures without breaking down
         • endogenous DNA polymerase = breaks down at temp below 95C.
   • Excess Nucleotides
     • A,G,C,T = building blocks of DNA molecules -DNA polymerase grabs it from liquid floating around it and attaches them to end of primer
   • Magnesium Chloride
     • Thermostable polymerase uses this as a cofactor
       • Role similar to catalyst - not consumed during reaction but reaction cannot proceed without
     • Too much:
       • Reaction will speed up which isn't good because then the polymerase will work too quickly and make errors in copying process
         • Therefore:
           • many different strand of different DNA produced that doesn't properly rep. the original sample
     • Too little:
       • reaction won't go as quickly if it should go at all - you won't get the amount of copies you intended
         • some of the polymerase won't be activated therefore won't work especially because the heat will have taken apart the DNA already = ruined experiment

2. Primer Design

   • Things to avoid:
     • long runs of a single or more bases
     • have a G or C at the 3' end
     • not complementary to other parts of the genome
     • should not anneal with themselves.
       • Primer dimer = Two primers bind together (ex. forward and backwards)
       • Hairpin loop = one primer binds on itself
   • Requirements:
     • Must have sequence complementary to target
     • 40-60% GC content
       • makes for a more stable primer because of the 3 hydrogen bonds between G+C instead of the 2 bonds between A+T
     • melting temperature between 55-65 deg.
       • Tm = 2(A+T) + 4(G+C)
     • Length between 18-30 nucleotides
       • longer the sequence the more specific. the shorter = less specific and therefore may bind to other parts of the genome
   • Adding Restriction sites
     • Required for cloning
       • you add the appropriate sequence to the 5' end of the primer
         • the mismatched region (restriction site) will not pair with the template b/c not complementary but each strand made becomes a template for the subsequent rounds of PCR
           • Therefore = the mismatched sequence will be copied into every subsequent product
             • the restriction enzymes require at least 3-4 bases on both sides of the recognition site in order to cut efficiently

3. Equipment

   • Thin walled tubes
     • Used because the thin walls produces more even and efficient heat transfer in the sample.
   • What is the function of the heated lid?
     • Prevents condensation on the lid.
       • Normal reaction = reaction heated and the water will evaporate and condense at the colder lid. = therefore [salt] will increase in the reaction and the polymerase won't work.
         • heated lid makes it so the water can't condensate because the cap is above boiling temperature so some water will evaporate but only enough to be a saturated as and will condense when the temperature cycles lower maintaining [liquid] and [salt]

4. Cycle 1

   • STEP 1: Initialization!
     • consists of heating reaction to 94-96C for 1-9 minutes
       • Or 98C if extremely thermostable polymerases used
       • Only required for polymerases that required heat activation my hot-start PCR
   • STEP 2: Denaturation
     • Heating the reaction to 95C for 20-30sec
       • Causes DNA melting by disrupting hydrogens bonds between complementary bases
         • produces ssDNA molc.
   • STEP 3; Annealing
     • temperature lowered to 60C for 30 seconds
       • allows annealing of primers to ssDNA template.
         • *usually annealing temperature only 3-5 deg. below melting temperature
           • if annealing temperature too low = non specific binding of primers with template - may even bind to each other = false product or no detectable product at all
         • polymerase binds to the primer-template hybrid and begins complementary strand formation.
   • STEP 4: extension/elongation
     • temperature used here depends on the polymerase used ~ Taq = 72 deg usually used for 1 minute
       • Taq synthesises new DNA strange comp. to template by adding free nucleotides to the end of the extending strand.
         • extension time depends on both polymerase used and length of template
           • Rule of thumb = 1000 bases/minute
             • Polymerisation of the new strand will continue either until the temperature rises again to start the new cycle
   • STEP 5: Final Elongation
     • occasionally performed at 70-74deg. for 5-15 mins. after past PCR cycle
       • ensures any remaining ssDNA fully extended
   • FINAL PRODUCT AT THE END:
     • two double stranded DNA molec for each you started out with
       • each new form strand has one end with a primer and the other free - as long as polymerisation was able to proceed during the extension time

5. Cycle 2

   • denaturation will create 4 single strand template molc
     • 2 from original template
     • 2 - newly synthesized
   • Annealing
     • primers and taq willl bind to each of the strands
       • Taq will bind and synthesise.

6. REACTION 101

   • ~30 cycles altogether -by then billions of copies
   • "Chain Reaction" - because replicated strands acts like templates for the subsequent strands = exponential copies made
   • all ingredients mixed and placed in tube
     • 'ready mixes' of ingredients can be purchased
       • benefit:smaller margin of error. ex. if ingredient stuck in pipette end - [wrong] therefore wrong