The Polymerase Chain Reaction Flashcards
What is needed for the reverse transcription of RNA into cDNA?
Reverse transcriptases from a range of RNA viruses require a small DNA primer and a target RNA strand.
These reverse transcriptases will then initiate the production of the DNA copy of the RNA.
As DNA polymerases cannot copy an RNA template this represents an essential step prior to PCR from an RNA source.
What is the purpose of using reverse transcription PCR?
Reverse transcription PCR enables the amplification of a specific messenger RNA from a sample for the purposes of its detection, cloning or quantification.
For quantitative measurements RT-PCR is usually performed as a quantitative PCR (Q-PCR) using real time measurements of the amplification process.
What is the major advantage of using quantitative PCR?
It allows for accurate estimation of the amount of target sequence within a sample.
Accurate comparisons between samples offers offers great benefit if variation in a chromosomal copy number or mRNA level has bearing on disease status.
What are the problems in estimating the amount of target sequence within a sample using conventional PCR?
Estimates using conventional PCR, where the amount of product in the reaction tube was observed at the end of a series of cycles, either by gel electrophoresis or other means, were subject to many problems.
For example, when the end point of the reaction is reached the PCR reaction itself becomes limited and reaches a non-exponential amplification phase or ‘amplification plateau’. At such points estimates of relative copy number are extremely unlikely to be accurate.
What methods were developed to get around the problems encountered in detecting the quantity of PCR product accurately?
Quantitative PCR methods that monitored the PCR reactions at each cycle were developed.
This got around the fact that by the linear and plateau phases of PCR the amount of PCR product no longer accurately reflects the number of target copies initially in the sample (and therefore could not be measured as a representation of quantity).
What is reverse transcription PCR?
Reverse transcription PCR describes the process whereby RNA from a cell is first converted into cDNA, which is then used as a template for exponential amplification using RT-PCR.
Most commonly the reverse transcription step is performed and then the sample is taken for PCR.
What do you need for PCR?
DNA template, DNA primers, dNTPs, DNAP, Buffer, Thermal cycler.
What are the main steps in the PCR reaction?
1) . Primary denaturation.
2) . Denaturation at 94 degrees Celsius where the dsDNA become ssDNA.
3) . Annealing of primers to ssDNA at 54 degrees Celsius.
4) . Extension phase at 72 degrees Celsius where the polymerase adds dNTPs from 5’ to 3’ to create two double strands.
5) . Final extension.
What are DNA primers?
Oligonucleotide sequences that deter in the specificity of DNA amplification.
For most PCR procedures you will require 2 primers of different sequences designed to anneal to complimentary strands of template DNA.
How would you go about designing a primer?
The first step is to determine the sequence of the region of the DNA of interest. Deciding the exact primer sequence you are going to use should always take into account:
1) . The length and base composition of the sequence.
2) . The melting temperature of the primer (Tm).
3) . The 3’ terminal sequence.
4) . The ability of the primers to base pair to themselves or other primers used in the reaction.
Primer sequences should be specific for sequences flanking the target region.
What is the optimum primer length?
18-25bp as it is long enough to give good specificity.
What should the base composition of a good primer be?
The base composition of primers should be roughly equal composition of each nucleotide and should have an overall GC content of 40-60%.
What types of sequences should be avoided when designing primers?
Repetitive stretches of the same nucleotide should be avoided as this can lead to slipping of the primer on the template and can reduce specificity. All four nucleotides should be evenly distributed.
What is the melting temp (Tm) of the DNA?
The Tm of the DNA is the midpoint on the transition between double stranded and single stranded DNA (where half the DNA is ds and half is ss).
What is the Tm of a primer?
The Tm of a primer is the temperature at which half the primers on the PCR mix are annealed to the target template creating double stranded stretches, while the other half are free in solution.
How can the Tm of a primer be calculated?
The Tm of primers up to 20nts long can be roughly calculated by adding the numbers of bases in the primer and applying the following formula:
Tm = [(number of C+G) x 4degreesC + (number of A+T) x 2degreesC]
This formula reflects the fact that the GC base pairs are more stable than AC base pairs due to their greater hydrogen bonding.
How is the annealing temperature (Tm) of the primer theoretically calculated?
The Ta of the primers is deemed to be 5degreesC less than the highest primer Tm in the primer pair.
A pair of primers with similar Tms should be chosen so that an annealing temperature that will work for both can be selected.
Although Tm and Ta calculated values are a useful indication of melting and annealing temperatures for the reaction, in practice is usually necessary to determine the optimum temperatures empirically.
What region of the primer sequence is the most critical for specific amplification and why?
The last 3 bases of the 3’ end of the primer is the mos critical for the specific amplification as this is the end that the polymerase extends from and should exactly match the target sequence.
When designing primers the 3’ end should be selected to avoid known polymorphic sequences.
What might happen if the 3’ end of a primer is able to base pair with itself or another primer on the reaction?
The 3’ end must not allow base pairing to itself or another primer in the reaction as this can lead to primer dimers. A primer dimer is the product of primer extension either on itself or the other primer in the pair.
As primer dimers contain one or both of the primer sequences and their complimentary sequence they provide excellent templates for further amplification. These smaller products are also amplified far more efficiently and sequester primer away from the intended target sequence.
What can happen it you have runs of 3 or more Gs or Cs at the 3’ end of a primer?
It can lead to mispriming in GC rich regions of the template DNA.
What should the final 3’ base of a primer ideally be?
The final 3’ base should ideally be a G or a C as they have the strongest complementarity and can aid specificity of annealing to the target sequence.
As and Ts should be avoided in the last few bases at the 3’ end of the primer.
Why should primers be designed to avoid regions of internal complementarity?
Inverted repeats and self complementarity sequences of more than 3bp may cause secondary loop structures where part of the primer has base paired with itself creating a double stranded region.
What software can help you design primers?
Primer 3 online,
Commercial software,
Can also run in silico PCR at UCSC genome browser.
Why must the DNAP used be thermostable?
DNAP must be thermostable in order to withstand the high denaturation temperatures. This enables repeated amplification cycles to take place with a single addition of enzyme when the reaction mixture is made.
Give 2 examples of DNA polymerase enzymes that may be used on PCR.
1) . Taq DNAP - works at 72-75C, lacks proofreading 3’-5’ but has very good processivity and is a good general purpose enzyme.
2) . Pfu DNAP - retains proofreading activity but has lower activity. Can be enhanced by mixing with thermostable factors such as Pfu turbo.
What are Hot Start enzymes?
Hot Start enzymes are inactive until the first denaturation step in the PCR cycle. This prevents any extension occurring at room temperature during PCR setup when primers may be bound nonspecifically to DNA.
Non specific products generated before exponential amplification during PCR will be amplified along with your desired product.
How can polymerases be improved?
By modification or mixing with other enzyme.
How are PCR products traditionally analysed?
Products are traditionally visualised using horizontal gel electrophoresis.
Agarose gel stained with ethidium bromide or sybr green.
The speed at which molecules pass through the pores on the gel depends on the size and charge of the molecules. Smaller DNA fragments will pass through the pores more quickly than larger ones. DNA will separate into bands across the gel according to size.
Describe some of the limitations of PCR.
If a large quantity of product is needed it might be better to clone the product.
The size of your product may have a bearing on the success of your PCR reaction. Standard PCR reactions are efficient on smaller fragments less than 2kb. Enzyme mixes for long range PCR fragments between 5-20kb are available.
PCR may be inhibited by a wide array of compounds derived from DNA samples or reagents used to extract the DNA template. Haemaglobin and heparin both inhibit PCR as do ethanol, SDS and proteinase K.
What elements of the PCR reaction require optimisation?
1) . DNAP choice
2) . Annealing temperature Ta
3) . Mg2+ concentration
4) . Primer to template ratio
5) . Additives
What might you want to consider regarding DNAP optimisation?
What is the best enzyme choice for your desired product?
Do you need 3’-5’ proof reading activity for a high fidelity product? Or a mixture for very long products?
Some enzyme modifications are designed to increase the specificity of the reaction. Hot start enzymes are inactivated by chemical modifications such as wax barriers or Taq directed antibodies. Inactivation prevents low levels of polymerase activity at room temperature from creating products resulting from non-specific primer binding. Such small fragments that may otherwise be produced are amplified efficiently and can reduce the amount of target DNA at the end of the reaction. Generally a prolonged incubation at 94C before the first cycle in the PCR reaction is enough to activate the hot start enzyme.
Why is optimising the annealing temperature (Ta) of the enzyme important? How can this be done?
If the Ta is too high then the reaction may fail to amplify product. If the Ta is too low then non-specific product will be produced.
The Ta may be optimised by setting up multiple identical reactions and using a thermocycler which can be set to produce a temperature gradient. This enables the simultaneous testing of various temperatures over a defined range.
A touchdown PCR technique may also be used. It initially starts with the Ta higher than the primer Tms and during the PCR reaction the Ta is lowered in a stepwise fashion to below the Tm. This ensures that specific primer binding occurs before any non-specific annealing. Reduces the possibility of forming non-specific products
