Polymerase Chain Reaction(PCR) is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified in rather simple manner, thanks to Kerry Mullis invention way back in 1983. Cell free DNA amplification can be performed in many different ways ,which can be used for identifying and manipulate DNA, detect infectious organisms, also helps in finding mutations, in human genes and numerous other tasks.
lets you pick the piece of DNA you are interested in and have as much of it as you want.–Kary Mullis
In the PCR method, a pair of primers is used to hybridize with the sample template DNA and define the region of the DNA that will be amplified. Primers are also referred to as oligonucleotides. To put things in simple words PCR is a technique where one single molecule of DNA (known as template) can be amplified many folds using short fragments of single stranded synthesized DNA, called as primers that bind to our template.
This process involves DNA polymerase enzyme along with components required for DNA synthesis ( Buffer for enzyme, Mgcl2, all four deoxy nucleotides- dNTPS: Adenine triphosphate , Thymine Triphosphate, Guanine triphosphate , Cytosine triphosphate ). DNA polymerase forms a vital component of PCR reaction and most often forms a deciding factor for efficiency of final DNA amplification.
Polymerases are enzymes have the ability to put together new strands of DNA in 5′ to 3′ direction of primer or towards 5′ direction of template, using the provide nucleotides and template DNA as reference. In older days, PCR reaction was considered as cumbersome because the high temperatures needed to denature the DNA and this would kill the polymerases activity. This meant that after every heating cycle, new polymerases needed to be manually added to the reaction– an expensive endeavor. However after discovery of DNA ploymerase from thermophilic bacteria sources, Thermus aquaticus (hence the common name for DNA polymerase used for PCR – Taq) allowed us to perform the reaction in one go , without the need of supplementing polymerase after each reaction. These modern day Taq comes with various desired features such as high speed, fidelity, processivity (ability to complete long reads sometimes even 30Kb or more)), and their ability to read GC rich templates. The list of improved taq’s are improving day by as day so keep always on look for DNA polymerase which perfectly fits your experiment.
All commercial DNA polymerase comes with optimal buffer (10x buffers and used at 1x concentration for PCR reactions) and all four dNTPs are available as one mix or brought separately and the working concentration should be around 2.5mM final volume for each PCR reaction. All the components (template DNA, primers, buffer 1x, dNTPs 2.5mM , DNA polymerase 1unit ) goes into a thermocycler which provides the required temperature necessary for different stages of PCR reaction.
A typical PCR reaction involves following stages :
Initial denaturation : This step is performed only once at the beginning of reaction and temperature is maintained around 94-95 C for 10 seconds. This allows template DNA strands to be separated and allows primers to find its targets in subsequent steps.
Denaturation : In this step, the reaction is heated to 94-98°C for 30 seconds. This step denatures your DNA and primers, which will allow them to anneal to each other in the next step. Generally PCR reactions are performed for 25-35 cycles ( depends on need of the entire experiment but generally 30 cycles is decent enough number beyond 35 cycles Taq tends to incorporate wrong nucleotides and generate mutations) and denaturation step is first step for all cycles followed by annealing and extension.
Annealing : This step allows primers to bind to specific region on template and generally performed at much lower temperature ( 50-60). Annealing temperature depends on melting temperature of primer pair and this temperatures should be enough to form strong base pairing between primer and template and also should prevent mismatch at other places on template DNA . The melting temperature (Tm) is the temperature at which one-half of a particular DNA duplex will dissociate and become single strand DNA. The stability of a primer-template DNA duplex can be measured by its Tm. Primers with melting temperatures in the range of 52-58°C generally produce better results than primers with lower melting temperatures. While the annealing temperature can go as high as 72°C, primers with melting temperatures above 65°C have a higher potential for secondary annealing.
Extension: This step is carried at 72 degree C and time of extension depends on the size of DNA fragment to be amplified and Taq used for the reaction. Some high end Taqs of modern day industries amplifies 1Kb of DNA in 10-15secs without any error. The steps denaturation, annealing and extension are repeated for 30-35 cycles before heading to final extension step. These additional cycles produce millions of exact copies of the target region of DNA. Because during the second cycle of this process, extension can occur on both the original copy of genomic DNA and the newest pieces (the red colored ones in the diagram) subsequent extensions are quickly limited precisely to the target region for which primers are designed.
Final extension: This step requires components of PCR to be at 72 degree C for 10 minutes, which allows the polymerases to finish reading whatever strand they are currently on and helps reduce the number of truncated copies in our final product.
Finally Pcr products can be stored at 4 degree C before progressing to next step using the PCR product , which can checked on gel and further by sequencing the PCR product.
For doing a successful PCR , the one step which is most important is that of designing oligo / Primer for the DNA to be amplified.
Few important points to remember for primer designing :
First thing is the length of primer ,usually 18-21 mer is optimal but sometimes one needs to include the restriction enzymes sites also to facilitate cloning.Then it turns out to be around 30. Usually they also work as efficiently as the smaller length primers, provided you take care of following things:
Presence of the secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction. the most famous among these is the Hairpin formation.Hairpin s is formed by intra molecular interaction within the primer and should be avoided at any cost or else your primer wont be able to find its target.The second common problem with longer primers is the ability to form self dimers (formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself.)
These two things can be taken care of by using various online web pages ,which provide you tools to cross check the oligos you designed for hairpin formation and self dimers. The best one according to me is from “Integrated DNA Technologies “
Secondly the annealing temperature
annealing,allows binding of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used.the melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.Generally the optimum range for annealing is 55°-65°c but usually desirable results can be obtained from 50°c. If sometimes you don’t get a clear band ,try doing a gradient PCR to find out exactly at what temperatures you see band of your choice.
Some tips for primer design :
* Base composition should be 50-60% (G+C)
* 3′-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product
* Primers should end (3′) in a G or C, or CG or GC: this increases efficiency of priming
* series of three or more Cs or Gs at the 3′-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.
Primer length and sequence are of critical importance in designing the parameters of a successful amplification. The melting temperature of a nucleic acid duplex increases both with its length, and with increasing GC content. A simple formula for calculation of the (Tm) is:
Tm = 4(G + C) + 2(A + T) °C
PCR Links :
Sites for designing primers :