By Dean A. Haycock

Special To BioWorld Today

If DNA is the word, polymerase chain reaction (PCR) is the printing press.

The DNA “word“ may lie in a speck of a murder victim’s blood, in a bone of an extinct animal or in the cells of people carrying a lethal disease. Whatever its source, scientists turn to PCR to amplify the message millions of times in just a few hours.

Like a printing press, PCR starts with a small message and, in an automated and highly specific way, churns out copy after copy.

Instead of ink, PCR uses a solution of specialized polymerase enzymes, which synthesize complementary strands of DNA. These are mixed with the DNA alphabet (A, T, C and G) and a couple of DNA fragments called primers, which flank and mark the target region. This test tube Gutenberg process is still revolutionizing the diagnosis of genetic diseases, forensic science, evolutionary biology, paleoarcheology, genetic diversity studies and clinical medicine.

But like a printing press, PCR techniques may be limited in the size of the editions they can print. The primers pick out specific regions of the DNA for replication. Amplifying an organism’s entire genome is another story. It requires specialized strategies. Many of these strategies for whole genome amplification still have not been fully evaluated.

In the Dec. 10 Proceedings of the National Academy of Sciences, however, Vivian Cheung, now a resident at the Children’s Hospital in Philadelphia, and Stanley Nelson, an assistant professor of pediatrics and biological chemistry at the University of California in Los Angeles, describe a method that may greatly enhance researchers’ ability to copy and analyze all of the genetic material in a small sample of DNA. In their paper, “Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA,“ Cheung and Nelson found that one type of PCR, called degenerate oligonucleotide primed-PCR (DOP-PCR), can be modified to amplify all of the PCR-based markers they have tested so far. Furthermore, they were able to accurately genotype all the markers from amplified human genomic DNA. This means that DOP-PCR can be used as a general method for amplifying small genomic DNA samples.

DOP-PCR itself was introduced several years ago and used to concentrate on a subset of DNA fragments which are useful as limited probes. It is not so useful, however, for researchers who want to be sure they are amplifying every fragment, every base pair, in genomic DNA. It is possible to amplify genomic DNA from a single cell with other versions of the PCR technique, but the number of tests possible with the resulting amplified DNA may be severely limited. The techniques do not allow vast amounts of amplification. It is possible to use up the DNA source before all tests are complete. So it is not the small size of the starting material that distinguishes the new modified technique from other PCR techniques; it is the yield in amplified genomic DNA.

“We demonstrated that with an amount of DNA less than a nanogram, we can amplify the whole genome a thousand fold,“ Nelson told BioWorld Today. The potential applications for the modified DOP-PCR technique are widespread. In forensic science, for example, fairly large amounts of DNA can be recovered from a dried blood spot, but the sample can be quite degraded. A degraded sample may leave little of the large genomic DNA that is required for an analysis. The modified DOP-PCR technique allows whole genomic amplification amidst the background of highly degraded DNA. The same applies to samples collected by paleoanthropologists.

The technique also could make it easier to accumulate DNA samples from individuals throughout the world, one of the goals of the human genome diversity project. Some people object to having their blood drawn for cultural reasons. And storing it presents logistical problems. Fortunately, there are other ways to obtain DNA. Blood can be spotted onto cards. Cells can be scraped from the inside of the mouth. Hairs, along with a small amount of tissue, can be plucked.

“We don’t know how much DNA we will want and we don’t know what type of tests people will want to do on them, but in all likelihood PCR-based tests will be the most commonly performed. If we can send people around the world plucking hairs or doing mouth washes, it will be easier than drawing blood samples,“ Nelson said.

Unlike blood samples, these methods do not allow researchers to immortalize the cells by growing them in a cell culture. But that would not be a problem with the new technique. In fact, by making it unnecessary to immortalize samples for future analysis, modified DOP-PCR might spare scientists the expense and trouble of preparing and maintaining cell cultures.

Of course, the new version of PCR could stretch the usefulness of DNA samples that have already been collected.

“There are probably millions of stored DNA samples that could be useful for analyzing the genetics of various diseases, but the amount of DNA you are going to get from them is limited. You could use it up very quickly [using other PCR techniques],“ Nelson said.

Future experiments in Nelson’s lab will push the limits of the modified DOP-PCR procedure to determine how long the amplification can be carried out and how small a sample of DNA will serve as sufficient starting material.

“It may be now that after you have attached the primer binding sites in an unbiased manner you can keep reproducing the genome indefinitely. We don’t know how long we can go with that,“ Nelson said.