PCR Analysis: An Examination

The polymerase chain reaction (PCR) is a powerful technique which uses repetitive cycles of primer annealing, primer extension, and product denaturing to produce an exponential increase in the copy number of the target DNA. Two primers are used, which flank the region of interest (See figure below). In the presence of a thermostable polymerase, the substrate DNA is denatured at 95°C, the primers are annealed at 50°C, and the polymerase generates new copies of the DNA at 72°C. In the next cycle, twice as many molecules are available for primers to anneal to. Thus for n cycles, 2n products are produced. Typically 30 cycles are used, which generates 230, or more than one billion, copies from one original. For a 500 bp fragment, this corresponds to about 1 pg of DNA from each copy, or a microgram of DNA from each femtogram of starting material.

One cycle of PCR
One cycle of the polymerase chain reaction (PCR). The double stranded substrate is denatured at 95°C. Primers are annealed to the single strands at 50°C, and extended at 72°C to produce two copies of the original template.

 

An important aspect of PCR is its ability to amplify specific sequences from complex mixtures. Given primers of sufficient specificity, and with some effort at optimizing conditions, a single sequence can be isolated from total RNA or genomic DNA using this technique. This allows rapid cloning of target sequences without creating and screening a library. The flanking sequences needed to design the primers can often be derived from end sequencing of proteins, from knowledge of gene structure (i.e. using the poly A of RNA as a priming site) or from conserved regions in homologous sequences.

Ideally, a PCR reaction gives one product of a predicted size, which needs only to be separated from the unused primers and dNTPs. In practice, particularly in amplifications from complex mixtures, multiple products are often produced.

One of the most common artifacts is “primer-dimer”. This is produced when the primers used are able to hybridize to each other at their 3' ends. These hybrids are extended efficiently into a 30-50 bp structure, which competes for amplification with the target DNA. The result is a low molecular weight band, which in the worst cases is over 90% of the reaction product.

Nontarget bands are also produced from mis-priming. If the primers have sufficient homology to some nontarget DNA region, this region will be amplified. It is important to realize that mismatches between primer and target only impede the first round of amplification.

Once the first product has been copied, a perfect match is generated for the next annealing (See figure below). Mis-priming is minimized by using the highest annealing temperature which gives product, and by optimizing the Mg+2 concentration in the reaction.

It is also important to avoid over-amplification. Once a product has accumulated to >1µg/reaction, the probability of one of the primers finding an unintended priming site within the target sequence is greatly increased. This generates a truncated fragment which is more efficiently amplified than the longer target. Since this smaller fragment contains a portion of the target sequence, it can interfere with subsequent analysis of the reaction such as sequencing or blot analysis.

Mis-priming in PCR
Mis-priming can produce unintended products. After a single round, any mismatches between the primer and the improper sequence will be eliminated, as the figure above shows. The improper target will have no impedance to its amplification along with the target DNA.

 

PCR Amplification


Protocols and conditions for PCR depend strongly on the enzyme, primers and substrate used. General guidelines for use of Taq polymerase are given below:

Reaction mixture:

  • 25-50µl of Taq Buffer
  • 0.2 mM dNTP's
  • 0.5 mM each primer
  • 2-3 units Taq Polymerase
  • Target sequence

Taq buffer contains 67mM Tris-HCl, pH 8.8, 1-5mM MgCl2, 150mM (NH4)2SO4, and 10mM BME. The concentration of Mg++ will vary from 1-5mM, depending upon primers and substrate. The amount of substrate used depends upon the concentration of target sequence in the sample DNA. The target will be amplified by up to one million times in a successful reaction, but the amplification will usually plateau at 1-10µg. Thus, 1 pg of target sequence in the reaction is a good place to begin.

Prepare the reaction in an 0.5 ml microcentrifuge tube and overlay it with 50 -100µl (1-2 drops) of mineral oil to prevent evaporation. Place the tube(s) in a thermocycler and run 20-30 amplification cycles.

CYCLE CONDITIONS:

Denature the reaction at 90-94°C for 0.5 - 1 minute. The time and temperature should both be the minimum compatible with product production. Taq polymerase has a half-life of 20 minutes at 94°C. Consequently, after 30 cycles with denaturation at 94° for 1 minute per cycle, more than half the enzyme activity will be lost.

After denaturing, anneal the primers at 45-60°C. This temperature is one of the most critical optimization parameters. Start at 5-10°C below the lowest calculated melting temperature (Tm) of the primer pair, and increase for subsequent reactions until yield begins to decline. The annealing step requires 0.5 - 1 minute.

Finally, increase the temperature to 72°C, the optimal temperature for Taq polymerase. Allow the Taq to extend the annealed primers for 1.5 - 3 minutes. Many programs increase the extension time for each successive cycle, to compensate for lost Taq activity and increased substrate concentrations.

CYCLE SUMMARY:

  1. 90 - 94° - 0.5 - 1 minute
  2. 45 - 60° - 0.5 - 1 minute
  3. 72° - 1.5 - 3 minutes
  4.  Return to (1)

Thirty such cycles are usually sufficient to amplify 1-10 µg of product. You may use 35-45 cycles, but internal priming on the product and over amplification of unwanted bands often result from over-cycling. Generally, it is better to focus on optimizing reaction conditions than to go beyond 35 cycles.

The PCR process is covered by patents owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche LTD.

 

NEXT TOPIC: PCR Analysis: Yield and Kinetics

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