The polymerase chain reaction detection and characterization of extremely small quantities of viral genomes or transcripts

The ability to characterize, work with, and control many viruses is limited by the fact that they are present in very small quantities in a given cell, tissue, or host. The use of a fluorescent stain such as ethidium bromide allows the ready detection of 100 ng or less of dsDNA. For a viral genome of, for example, 30,000 base pairs, this works out to be approximately 5 X 1011 molecules. Radioactive labeling can greatly increase the sensitivity of detection, but it is not always possible to specifically label the DNA fragment of interest in the tissue being studied.

The problem of visualizing and manipulating extremely small quantities of DNA was overcome in larger part by developments of the polymerase chain reaction (PCR) initiated and commercialized by scientists at the Cetus Corporation in the mid-1980s. The principle, illustrated in Fig. 11.11(a), is quite simple. Consider a fragment of dsDNA present as even a single copy in a cell or animal. If this DNA is denatured by heating above its denaturation temperature and short oligonucleotide primers can be found to anneal to the opposite strands at positions not too far away from each other (e.g., within a thousand bases or so), two strands of cDNA can be synthesized using DNA polymerase. The new product will be double stranded in the presence of the nonprimed denatured DNA.

Now, if the newly synthesized dsDNA is itself denatured, and the priming and DNA synthesis step is repeated, this short stretch of DNA will be amplified as compared to the strands of DNA that did not bind primer. This process can be repeated many times in a chain reaction to amplify the desired strand of DNA to useful amounts.

To work properly, the oligonucleotide primers must be long enough to be highly specific, but short enough to allow frequent priming. The appropriate length works out to be about 20—30 bases. The technology for synthesis of 20- to 30-base oligodeoxynucleotides is well established and can be chemically performed relatively inexpensively. Indeed, numerous large and small biotechnology companies make oligonucleotides commercially.

Also important is the ability to do the reaction, denaturation, and reannealing in a single tube many times over. This is accomplished by using the heat-stable DNA polymerases isolated from organisms such as Thermophilis aquaticus (Taq), which live in hot springs, and the use of computer-controlled thermal cyclers that can repeat the annealing, synthesis, and denaturation steps rapidly and repeatedly over 1—4 hours.

(a) Original DNA

Denature

PCR primer 1 PCR primer 2

Approximate number of molecules amplified

"i3

Synthesize new DNA

Uninfected Latently infected ganglion HsV ganglion

I control I Size std

Denature and make new DNA

Actin -

Repeat n times

B7 bp

Exponentially amplified copies of DNA between primers

Fig. 11.11 Amplification of DNA with the polymerase chain reaction (PCR). (a) The basic method requires specific primer sets that can anneal to opposite strands of the DNA of interest at sites relatively close to each other. After denaturation, the primers are annealed, and DNA is then synthesized from them. All other DNA in the sample will not serve as a template. Following synthesis, the reaction products are denatured, and more primer is annealed and the process repeated for a number of cycles. The use of heat-stable DNA polymerase allows the reaction to be cycled many times in the same tube. A single copy of a DNA segment of interest could be amplified to 109 copies in 30 cycles of amplification. Can you demonstrate this mathematically? (b) The amplified DNA products from a segment of HSV DNA. A total of 1 jig of nonspecific DNA was added to each of a series of tubes, and viral DNA corresponding to the copy numbers shown was added. Following this, primers, heat-stable DNA polymerase, and nucleoside triphosphates were added, and 30 cycles of amplification were carried out in an automated machine. The reaction products were fractionated on a denaturing gel and visualized by autoradiography. The asterisk denotes a longer exposure of the products of the two most dilute samples. The lower gel shows the results of amplification under identical conditions of DNA isolated from two rabbit trigeminal ganglia. One was taken from a control rabbit, and the other was taken from a rabbit that had been infected in the eye with HSV followed by establishment of a latent infection. The use of rabbits to establish HSV latency is shown in Fig. 17.10. Amplified DNA from each sample was fractionated in the lanes shown; in addition to the amplification products, a sample with PCR-amplified HSV DNA as a standard (std) as well as some size markers were fractionated.

In practice, the method can be used to detect the presence of extremely small amounts (less than a single copy/cell) of a known viral genome by selection of appropriate primer pairs based on the knowledge of the sequence of the genome. An example of the use of PCR to detect HSV genomes is illustrated in Fig. 11.11(b).

PCR can also be used to look for the presence of genes related to a known gene. Such detection is based on the assumption that regions of a DNA sequence encoding a gene related to the one in hand will contain some stretches of identical or highly homologous sequences in their genomes. Detection can be accomplished by amplifying the DNA in question with a series of potential primer sets. If one or several of these yield products of a size within the range of those seen with the known gene, these products can be isolated and sequenced. If necessary, this can be done after the amplified fragment or fragments of interest are cloned using methods outlined in Chapter 22, Part V.

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