PCR Flashcards
issues with molecular cloning
-There might not be enough DNA.
-DNA might be mixed in with lots of other DNA molecules.
-No convenient restriction sites
-The gene we are interested in:
=Might be a few thousand bp out of several billion bp.
=May only be present in few copies.
=Won’t have enough convenient restriction sites.
what does PCR stand for
-polymerase chain reaction
what does PCR do in regards to DNA replication
-amplifies a specific piece of 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.
-PCR is basically DNA replication in a tube.
what is needed when making a new stand of DNA
-complimentary nucleotide base pairings
-double-stranded
-antiparallel
-semi-conservative
-Need to unwind DNA
-Need a primer
-Polymerase enzyme
-New nucleotides
how can we amplify one piece of DNA
-DNA is double-stranded and anti-parallel
-One cycle of PCR = dissociate the two strands and copy both 2 molecules of DNA
-Next cycle, do the same again
how can we amplify one molecules of DNA
-After one cycle, we have doubled this.
-Each cycle leads to doubling of DNA molecules = exponential
-After each cycle – molecules of DNA = multiply by 2 to the power of the number of cycles
why is Amplification during PCR exponential
-Molecules of DNA doubles in each cycle
-30 cycles of PCR is typical
-× 230 molecules of DNA
- > 1 × 109 (1 billion) molecules of DNA
-Exponential means that the number of DNA molecules doubles each cycle (linear would be add 2 for each cycle)
-Amplification is 2 to the power of number of PCR cycles
-Start with 1 piece of DNA => Get one billion
what are the 3 stages of PCR
-denaturation
-primer annealing
-primer extension
what happens during denaturation
-Double-stranded DNA dissociates into single-stranded DNA.
-at 95 degrees c
what happens during primer annealing
-Primers bind to complementary sequence on ssDNA. Primer binding is antiparallel.
-happens at 55-65 degrees c
what happens during primer extension
-DNA polymerase synthesises new strands of DNA from the 3’ end of the primers.
-happens at 68-72 degrees c
What do we need for PCR and why
-Template – DNA that we want to amplify
-DNA polymerase – copies DNA
-Primers – DNA polymerases need a free 3’-OH to start. Provided by a primer. (Therefore, we need to know a bit about our sequence)
-Deoxyribonucleoside triphosphates (dNTPs) – DNA bases to make the new DNA strand
-Buffer – Correct pH and ions (MgCl2)
-Thermocycler – maintains appropriate temperature for each stage of a cycle
-Way to maintain the correct conditions (pH, ions, temperature)
what’s Protein stability during PCR like
-Cycle goes through some high temperatures- 95C.
-DNA polymerases are proteins – usually destroyed by these temperatures
-Very first PCR (Kary Mullis, 1983) – added more polymerase during each cycle
-Nowadays, we have identified thermostable polymerases from thermophilic organisms
what’s taq
-DNA polymerase used in PCR
-Taq was the first discovered. Taq and Pfu are amongst the most common and widely used. Each lab has their favourite polymerase
-Different advantages/disadvantages – choice is somewhat dependent on application – pros and cons for each
-Thermostability is important
-Extension rate – how fast it can replicate a template
-Processivity – how often it falls off and has to re-associate
-Proof-reading and fidelity – contribute to accuracy. Pfu introduces fewer mutations into its PCR products
-different polymerases have diff features – choice is often dependent on application
what are some Important features of PCR primers
-Minimum size for specificity- 17 bp is an absolute minimum.
-Usually around 20 bp.
-Specific to your template- Primer sequence is complementary
-Primer binds in antiparallel fashion
-Come in pairs- Bind opposite strands in opposite orientation
-Appropriate melting temperature- Specificity
how do primers work in pairs
-Obviously have to be complementary to the sequence you want to amplify
-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)
-So will amplify region of DNA in between the primers
-Imagine you only have one primer
Only one strand is replicated => one dsDNA and one ssDNA
why is Melting temperature (Tm) important
-Temperature at which the primer will dissociate from the DNA template.
-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 the Tm.
-If it’s too high, tend to self-anneal
-If it’s too low, primer is not very specific
what’s Tm and annealing temperature (Ta) like
-Ta is just right: primers will bind to your specific sequence
-Ta is too low: primers may bind non-specifically to other DNA sequences.
-Ta is too high: primers may not bind efficiently (or at all), reducing product yield.
-Most of the primer is bound
-Bound correctly
how do you calculate primer Tm
-More complicated calculation
-Ability to self-anneal (secondary structure)
-Complementarity between primer pairs
-Repeated nucleotides
-3’ end stability
what are the problems with cloning
-No convenient restriction sites-Genes don’t usually have restriction sites exactly where you want them
-Not always directional-We often want cloning to be directional
-Might not have enough DNA-DNA often is present in tiny amounts
-DNA might be mixed in with lots of other DNA molecules-The DNA you want to clone is often part of a mixed sample
how can you ligate a PCR product directly into a vector
-PCR products have no 5’ phosphate
=Make primers with phosphates
=Add a 5’ phosphate (T4 PNK)
=Rely on the 5’ phosphate in the vector
-Taq adds a 3’ A overhang to its PCR products
=Remove the 3’ overhang
=Use clever vectors
=Use a different enzyme
-Blunt-ended cloning is not very efficient and is not directional.
what are the problems with ligating a PCR product directly into the vector
-Minor problems
=Phosphates – can be circumvented
=Taq – can remove A overhang / clever vectors that use the A overhang in cloning
-More pressing problems:
=Inefficient – have to screen a lot
=Half the time, the insert will be in the wrong orientation
how are restriction sites incorporated into primers
-3’ end of primer is the business end – cannot mess with this
-Can add pretty much what you like to the 5’ end
-RE on forward primer = EcoRI
-RE on reverse primer = BamHI (read from 5’-3’)
-The primer sequence is not complementary to the template and doesn’t bind
-Now we have a PCR product (a piece of DNA) that is our specific gene sequence and has restriction sites at each end, exactly where we want them
advantages of incorporated restriction sets into primers
-More efficient ligation: Sticky ends after restriction digest,
Vector can’t ligate to itself (digested with two enzymes), No need for phosphatase treatment
-Directional: Use different enzymes
what are the uses and steps of RT-PCR
-steps:
1) RNA is reverse transcribed into DNA (copy / complementary DNA = cDNA)
2) PCR is used to amplify a specific cDNA sequence
-uses:
=Molecular cloning – sometimes we want a cDNA sequence, rather than the whole genomic sequence (protein expression).
=Template for PCR is often cDNA, rather than genomic DNA.
=RNA expression
what happens in the first step of RT-PCR- cDNA synthesis
-First strand synthesis by a RT (often viral). Needs a primer, like other polymerases.
-Poly(dT) priming is common (mRNA). Can also use random primers
-RT synthesis first strand then loops back on itself a bit and starts another strand – forms a short hairpin
-Get rid of the RNA (often a nuclease)
-Second strand synthesis by -DNA polymerase (Klenow – retains useful functions but has lost 5’-3’ exonuclease activity)
-Primed by hairpin from RT
-Get rid of DNA loop
-Now we have cDNA
what happens during step 2 of RT-PCR- PCR
-Once cDNA synthesis is complete, step 2 of RT-PCR is a straightforward PCR reaction
-Template = cDNA from step 1
-Just need the usual reagents: primers, dNTPs, DNA polymerase
-And a thermocycler
how is Amplification during PCR exponential
-After a period of time, the building blocks (dNTPs, primers) run out
-DNA polymerase can also lose activity
how can you measure qPCR product
1)Fluorescent dye:
=SYBR Green
=Fluoresces when it binds
=Fluorescence is proportional to amount of dsDNA
=Not sequence specific
-Will also measure background / non-specific products
-Can only measure one thing at a time
2)Fluorescent probes:
=Sequence specific
=Can multiplex (several targets in one reaction – each one has a different coloured probe)
=Fluoresces when displaced from template
what does qPCR data tell us
-Both methods measure fluorescence (increases over time)
-Ct (cycle threshold) = the point at which fluorescence exceeds background levels
-Difference between Ct values is a relative measure of which sample had most template to start off with
what does a Low CT value mean
-fewer cycles of PCR needed to exceed threshold = more template at start
Calculating relative template amounts: ΔCt
-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 = 25 = 32
limitations of Calculating relative template amounts
-Assumes that reaction is 100% efficient and product doubles each cycle (not true!)
-Alternatives exist (machines will do it for you – take into account efficiency of reaction)
-it assumes that you have equal amounts of sample to start off with
how can we use the equation change in ΔΔCt
-ΔΔCt = ΔCtA − ΔCtB
-ΔCtA = CtAtest − CtAref
-ΔCtB = CtBtest − CtBref
whats the confusion between RT-PCR and qPCR
-qPCR is often referred to as Real-Time PCR, causing some confusion with RT-PCR
-Some overlap: qPCR is frequently RT-PCR, as it’s used for analysing mRNA levels.
-But it doesn’t have to be – possible to perform qPCR on templates other than RNA- Pathogen detection
-And we can perform RT-PCR that is not quantitative- Molecular cloning
