BCH 4053 Biochemistry IFall 2001
Dr. Michael Blaber
Polymerase chain reaction
- PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.
- PCR amplification is achieved by using oligonucleotide primers.
- These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.
- The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template.
- This results in the synthesis of new DNA strands which are complementary to the parent template strands.
- These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.
- The oligonucleotide directed synthesis of daughter DNA strands can be repeated if the new duplex is denatured (by heating) and additional primers are allowed to anneal (by cooling to an appropriate temperature).
The steps of:
- Template denaturation
- Primer annealing
- Primer extension
comprise a single "cycle" in the PCR amplification methodology.
After each cycle the newly synthesized DNA strands can serve as templates in the next cycle.
Note that half of the newly synthesized strands from the second round of replication have 5' and 3' termini which are defined by the annealing location of the primer oligonucleotides.
Summary of products at the end of each PCR cycle:
- The total of each of the different types of oligonucleotide fragments for each cycle can be summarized as follows:
- The amplification of the fragments follows the following pattern:
(2n - (n + 1)) x copies of each fragment of defined length (i.e. each end defined by the two PCR primers).
- There is always 1x copy of each of the original templates - the PCR as outlined never reproduces the full length template
- There will be 'n'x copies of each fragment with an indeterminate length (where 'n' is the number of cycles). The fragments of indeterminate length have one end defined by a PCR primer and the other end is of indeterminate length
- There will be
- For example, after 6 cycles we will have
- 1 x copy of each original template
- 6 x copies of each fragment of indeterminant length
- (64 - (6 + 1)) = 57 x copies of each fragment of defined length
- In the above case, the desired PCR product will be a duplex of the defined length fragment. The question is: how many will be produced?
- The original templates will not necessarily anneal with one another (there are more opportunities to anneal with other fragments that are present at higher concentrations)
- Likewise, the templates of indeterminant length will not necessarily anneal with one another
- Furthermore, as the number of cycles proceeds, the defined length fragments far outnumber the others
- Therefore, the original template, and fragments of indeterminant length are most likely going to hybridize with defined length fragments. This will "poison" our yield of desired duplex DNA with the length defined by the PCR primers
- Our expected amplification of the desired DNA after 6 cycles would therefore be:
57 - (6 + 1) = 50 x copies of each defined length fragments (i.e. 50 x duplexes of defined length)
where (6 + 1) represents the other products that can form a duplex with an undefined 3' end
The expected amplification of the desired defined length product with respect to the original template concentration 'x' can thus be represented by the formula:
[(2n - (n + 1)) - (n + 1)] x
(2n - 2(n + 1))
(this is often abbreviated to a simple rule of thumb for the amplification: (2n - 2n) x)
- The interpretation of this formula is that
- For a given number of cycles 'n' we make '2n x' total possible duplexes
- For a given number of cycles there will be '2(n+1) (or 2n in our approximation) x' duplexes which are formed from either the original template, or a fragment of indeterminate length, along with a fragment of defined length (and represent an undesired product)
- Thus, the total concentration of desired product (duplexes with a length defined by the PCR primers) will be
(2n - 2(n+1)) x (where x is the concentration of the original duplex)
- The theoretical amplification value is never achieved in practice. Several factors prevent this from occuring, including:
- Competition of complementary daughter strands with primers for reannealing (i.e. two daughter strands reannealing results in no amplification).
- Loss of enzyme activity due to thermal denaturation, especially in the later cycles
- Even without thermal denaturation, the amount of enzyme becomes limiting due to molar target excess in later cycles (i.e. after 25 - 30 cycles too many primers need extending)
- Possible second site primer annealing and non-productive priming
PCR was invented in 1985 by Kary Mullis, working for Cetus corporation somewhere near Berkeley, California. The original method of PCR used the Klenow fragment of E. coli DNA polymerase I. This enzyme, however, denatures at temperatures lower than that required to denature most template duplexes. Thus, after each cycle, fresh enzyme had to be added to the reaction. This was quite tedious. In addition to this problem with the enzyme, the samples had to be moved from one temperature bath to another to allow the individual steps of denaturation, annealing and polymerization (which all required different temperatures). This was pretty tedious too.
Thus, two main advances allowed the process to be automated, these advances were:
- The use of thermostable DNA polymerases, which resisted denaturation (inactivation) at high temperatures. Thus, an initial aliquot of polymerase could last throughout the numerous cycles of the protocol. The first thermostable DNA polymerase to be used was isolated from the bacterium Thermus aquaticus. It was isolated from a hot spring in Yellowstone National Park where it lived happily (i.e. it replicated its DNA) at temperatures in excess of 85 °C
- The development of temperature baths which could shift their temperatures up and down rapidly and in an automated programmed manner. These are known as thermal cyclers or PCR machines.
These two developments let to the automation of PCR. The PCR process is covered by patents owned by Hoffmann-La Roche Inc. (a faceless conglomerate you can trust).
Thermal cycling parameters
- The thermal cycling parameters are critical to a successful PCR experiment. The important steps in each cycles of PCR include:
denaturation of template
annealing of primers
extension of the primers
A representative temperature profile for each cycle might look like the following:
The initial denaturation of template is accomplished at 95-100 °C.
- Supercoiled plasmids are tougher to melt and may require boiling for several minutes, or may be initially denatured by using base (NaOH, followed by pH neutralization).
- Denaturation during the PCR experiment (i.e. second cycle onward) is usually accomplished at temperatures of 92-95 °C (usually empirically determined).
Primer annealing temperature
- Primer annealing temperature is an important parameter in the success of the PCR experiment.
- The annealing temperature is characteristic for each oligonucleotide:
- it is a function of the length and base composition of the primer as well as the ionic strength of the reaction buffer.
- Estimates of the annealing temperature can be calculated in several different ways.
- These calculated annealing temperatures are a starting point for the PCR experiment, but ideal annealing temperatures are determined empirically.
- Primer extension is usually performed at 72 °C, or the optimum temperature of the DNA polymerase.
- The length of time of the primer extension steps can be increased if the region of DNA to be amplified is long, however, for the majority of PCR experiments an extension time of 2 minutes is sufficient to get complete extension.
Number of cycles
- The number of cycles is usually between 25 and 35.
- More cycles mean a greater yield of product.
- However, with increasing number of cycles the greater the probability of generating various artifacts (e.g. mispriming products).
- It is unusual to find procedures which have more than 40 cycles.
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© 2001 Dr. Michael Blaber