PCR

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

Introduction
PCR
Colony PCR
About primers
Primer-dimers
Hot start PCR
Setting up PCR

About the PCR machine
 

Introduction

In the Simple Cloning Lab, PCR is used for 2 reasons:

  • To generate a high amount of DNA containing a particular region of the Lambda DNA. This is done by PCR – amplifying the Lambda insert of the pGTλ clone harbouring a recombinant plasmid.
    After labelling, the PCR products can be used as probes for Southern hybridisation.
  • To find out the size of the Lambda insert in pGTλ clones: the size of the PCR product and the insert size are virtually the same

example: to discriminate colonies harbouring the recombinant plasmid pGTλ3758 from those harbouring pGTλ3758ΔH, PCR with standard Gene Tech primers (see SCLResources>Sequences) can be used.

In both cases primers specific for the pGT4 vector sequence are used. These “standard Gene Tech PCR primers” anneal very close to the BamHI and EcoRI insert sites.

 

The Polymerase Chain Reaction

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The polymerase chain reaction (PCR) is an in vitro method for the enzymatic synthesis of specific pieces of (target) DNA. It is a rapid and simple means of producing (up to) mg amounts of DNA from minute quantities of target (“DNA amplification by PCR”).

The reaction requires:

  • two oligonucleotide primers that hybridize to opposite strands of the target DNA and flank the region to be amplified
  • a suitable DNA polymerase
  • the four deoxyribonucleoside triphosphates (dNTPs)
  • Mg2+ ions

To perform a PCR amplification, a mixture containing the target DNA, primers, dNTPs, and a heat-stable DNA polymerase is heated to 90-95°C to denature the strands of the target DNA. The solution is cooled to a temperature that allows the primers (single-stranded DNA molecules of about 18 to 30 nucleotides long) to anneal to their complementary sequence on the target DNA and provide the 3'-OH required for DNA synthesis. Subsequently, the DNA polymerase synthesizes a new DNA strand complementary to the target by extending the primer.

The polymerase used should be heat stable to tolerate the high temperature denaturation steps of all reaction cycles. Such an enzyme can be isolated from thermophilic bacteria like Thermus aquaticus. The enzyme from this bacterium is most frequently used in PCR. It is called Taq polymerase and has an optimal activity at 72°C.

This thermal cycling scheme of -denaturing/primer annealing/ primer extension- is repeated numerous times with the DNA synthesized during the previous cycles serving as a template for each subsequent cycle.  The result is a doubling of the target DNA present with each cycle.  This exponential accumulation of DNA sequences can theoretically produce over a millionfold amplification of the target in 20 cycles.  In practice, the amplification efficiency is less than 100%, and 20 to 40 cycles are commonly done.

The PCR is performed in a small volume (20-100μl), in small 200μl tubes, or in multiwell plates. A heating block with an automatic thermal cycler is used for precise temperature control.

As little as one molecule of starting template can be enough for amplification by PCR, because of the extreme amplification achievable. Therefore, any source of DNA that provides one or more target molecules can in principle be used as a template for PCR.  This includes DNA prepared from blood, sperm or any other tissue, from older forensic specimens, from ancient biological samples or in the laboratory from bacterial colonies or phage plaques as well as purified DNA.


New applications of the PCR are being developed constantly.
Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.
Protocols for PCR vary considerably for particular applications.

Colony PCR

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In the laboratory, “colony PCR” is often done: the reaction mixture is set-up using intact bacteria picked from a colony on an agar plate, rather than purified template DNA. The first denaturing step results in release of the DNA from the (lysed) bacteria. Then the DNA is available for annealing of the primers.

About primers

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Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature Tm of a DNA duplex increases both with its length, and with increasing (G+C) content:

a simple formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T)oC.

This formula could be used for primers smaller than 18 nucleotides
((G + C) means the number of C's plus the numbers of G's in the primer sequence).

A more accurate calculation of the Tm is according to the so-called Nearest Neighbour methode. you could use the Oligonucleotide Properties Calculator at http://www.unc.edu/~cail/biotool/oligo/.

This type of calculation must be used for primers of 18 nucleotides and longer.

Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One should aim at using an annealing temperature Ta not more than 5oC below the lowest Tm of the pair of primers to be used. Often, primers are designed to have a Tm of about 60 oC

One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated: this can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3'-most base is paired with a target. 
Read also about Hot Start PCR to avoid this.

A simple set of rules for primer sequence design is as follows

  • primers should be 17-30 bases in length;
  • base composition should be 50-60% (G+C);
  • primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
  • Tms between 55-80oC are preferred;
  • runs 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;
  • 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer-dimers can be synthesised preferentially to any other product;
    In the picture below the 3' end of primer A is complementary to the 3' end of primer B.
  • primer self-complementarity (ability to form secondary structures such as hairpins) should be avoided. See primer C  in the picture below.

 

Primer-dimers

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Primer-dimers are caused when two primers with the same or different sequences hybridise. Usually this is found in PCR reactions where the forward and reverse PCR primers hybridise. This appears as a 25-60 base pair long product. In the picture below, primer-dimers formed by primers A and B are shown; the newly synthesized DNA in red.

Hot Start PCR

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Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation. By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield.

PCR or polymerase chain reaction uses DNA polymerases which were isolated from thermostable organisms. Unfortunately, these thermophilic DNA polymerases show a very small but measurable polymerase activity at room temperature during assembly of the experiment.
This in-efficient DNA polymerase activity will catalyze the extension of any annealed 3' ends (so not only of 3’ ends of primers annealed at their specific fully complementary target site..). After PCR amplification, the resulting product contains a mixture of specific and non-specific bands.

This problem can be largely avoided by a very simple way: set up the PCR experiment on ice, and wait untill the heating block is over 90°C, before quickly transferring the PCR reaction tubes directly from ice into the block.
This is the simplest way of performing a HotStart.
Better protocols for Hot Start PCR (i.e. absolutely no DNA polymerase activity during set up of the PCR reaction) include chemical modifications of the polymerase, wax-barrier methods, and inhibition by an antibody against the heat-stable DNA polymerase.

 

Setting-up PCR reactions

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PCR can amplify very small amounts (e.g. a single copy) of template DNA.
So, very small amounts of contaminating DNA can give unexpected PCR products.

Often, the setting-up of a PCR includes the preparation of a so-called MasterMix, which is a large volume of a mixture containing all or most of the PCR reaction components (buffer, dNTP's, primers, polymerase), but without the DNA template
. Addition of the same mastermix to a series of different template-containing samples makes it possible to accurately compare the results of the samples.
One of the PCR reactions should be done without any template. Instead of a template-containing sample, just water is used. This control PCR is often called the water control.
It is very important to exclude in this way the presence of any template DNA in (one of the components of) the mastermix.
When the mastermix is added to template-containing samples which are already in the PCR tubes, it is extremely important to use a fresh pipet tip for each addition!!

 

About the PCR machine

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The picture above is typically shown in the display of a PCR machine, during (setting-up) a PCR run. You should read it like this:
There is a first denaturation step of 5 minutes at 94°C, followed by 25 identical cycles, with in each cycle a denaturation step of 30 seconds at 94°C, a primer annealing step of 30 seconds at 55°C and a primer elongation (by the polymerase) step of 30 seconds at 72°C. Finally a 7.5 minutes elongation step at 72°C has been programmed, followed by cooling to 4°C until the user stops the run manually.