Real time PCR for precise quantitative measures of viral DNA

In addition to its value for detecting vanishingly small amounts of viral genomes, PCR can also be used to make extremely precise quantitative measures of the amounts of viral genomes or transcripts present in different tissues, or under different conditions of infection. The amount of product formed in the PCR reaction is a function of a number of factors, but the most critical is the amount of target sequence available to begin the reaction in the first place. This follows from the dynamics of the rate of product formation where there will be a limited period of time when there is an exponential rate of accumulation of the product as the number of cycles increase until the available number of primers falls to a lower level where the rate of product formations becomes linear and finally reaches a plateau. Since with standard PCR methods the reaction is carried out for a set number of cycles, the amount of product formed will reflect the amount of target originally present only if the synthesis of PCR product is still in the linear range at the time of the last PCR cycle. Because total product formation is the endpoint, very rare sequences may only be amplified to a low level, moderately abundant sequences amplified to a level more or less proportional to their initial concentrations, and more abundant sequences will have reached a plateau in product formation relatively early in the course of the amplification. Thus, quantitative estimates of the amount of material present in the original sample can be difficult.

One way to overcome these reciprocity problems is to use a series of dilutions of the original sample for the amplification along with appropriate standards as shown in Fig. 11.11(b) where a series of dilutions of a fragment of HSV DNA corresponding to the copy numbers shown were carried out and subjected to PCR amplification. The gel shown was used to fractionate the reaction products that were made radioactive by the addition of a small amount of radiolabeled nucleoside triphosphate to the reaction mix. An amplified signal from 1000 copies of the genome provided a detectable signal with a short exposure of the gel to x-ray film.

The technique of real time PCR provides a much more reliable and precise method of quantitatively measuring the products of PCR reactions. This is accomplished by measuring the formation of the PCR products continually throughout all cycles of annealing and chain elongation. This is accomplished by using primers that contain a fluorescent marker that is only detectable upon the formation of the amplified product. Such primers usually have a fluorescent tag (a fluor) that is quenched either by the secondary structure of the primer or by a second ligand (the quench) attached to the primer. When the primer is not annealed to a DNA product, illumination of the reaction mix with a laser or other suitable light source will yield no fluorescence, but when the primer is annealed to the target or the amplified strand of DNA, it then is able to generate a signal which can be quantitatively measured upon illumination. Different fluors, each fluorescing at a specific wavelength, can be incorporated into different primers so that the rate of formation of several products can be simultaneously measured in the same reaction mix. The quantitative analysis of the human globin gene in peripheral blood macrophage DNA is shown in Fig. 11.12.

Nucleic acid from as little as a single cell can be subjected to PCR. The quantitative measure of viral genomes as a function of disease state or state of infection is vital for understanding the replication of HIV and its pathogenesis leading to AIDS. In the laboratory, PCR has also been very useful in studying the latent phase of infection of herpesviruses. Depending upon the details of infection and the exact strain of virus used, it has been determined that a typical

28 32 Cycle

Fig. 11.12 Real time PCR amplification of globin DNA in blood macrophages. Five-fold dilutions of DNA from these cells were subjected to multiple cycles of PCR amplification under conditions where the amplified DNA can be measured by measuring fluorescence. As the dilutions increase, the range of cycles in which the amplified signal is logarithmic also increases and, thus, the quantitative measure of the numbers of genes present decreases.

latently infected neuron in an experimentally infected rabbit might harbor between 10 and 100 viral genomes.

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