PCR and designing primers
Polymerase Chain Reaction 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’re interested in and have as much of it as you want”.–Kary Mullis
Different variants of pcr one can perform :
*Alu-PCR *Asymmetric PCR *Colony PCR
*DD-PCR *Degenerate PCR *Hot-start
*In situ PCR *Inverse PCR *Long-PCR
*Multiplex PCR *Nested PCR *PCR-ELISA
*PCR-RFLP *PCR-SSCP *QC-PCR
*RACE *RAPD *Real-Time PCR
*Rep-PCR *RT-PCR *TAIL-PCR
*Touchdown PCR *Vectorette PCR
PCR involves the following three steps: denaturation, annealing and extension.
First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then allowed to anneal to the complementary regions of the single stranded molecules. Finally, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94oC, 50oC and 72oC respectively.
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 annealing temperature
annealing,allowsbinding 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 synthesised 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.
PCR Links :
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PCR station
PCR links
Sites for designing primers :
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