EXQUISITE DETECTION

 

A single molecule can be detected because that molecule can be replicated exponentially until sufficiently many copies exist that they can be detected` macroscopically' (for example on a gel).But is it possible to exquisitely detect a substance that is not replicatable? The answer is yes. We have been exploring this possibility over the last two years.Our current `testbed' substance is reverse transcriptase (RT).We have chosen RT partly because its exquisite detection would potentially be of immediate use in HIV research. But on a more fundamental level, any of a number of possible substances would have served our primary goal of gaining insight into the process of exquisite detection and the most promising approaches to it.

In our current method, RT is incubated with a `paternal' strand of RNA and a `maternal' strand of DNA that partially overlaps it and acts as a primer for RT. If RT is present, the maternal strand is extended on the paternal template to produce a `daughter' strand. Thus, the daughter strand becomes a surrogate for the RT; its presence indicates the presence of RT. In fact, a single RT molecule can create multiple daughter strands. Hence, it acts as a `pre-amplifier' for a subsequent PCR amplification using primers specific to the daughter strand. This appears straight forward enough, but numerous problems arise in order to avoid false positives. If steps are not taken, then even without RT, a PCR reaction with our paternal and maternal strands will give a positive result, since all polymerases we have tried have sufficient RT activity to produce daughter strands.

Our assay for RT is approximately 100,000 times more sensitive than commercially available assays.We can detect 6 molecules (approximately 1 zeptogram) of RT in 10 microliters of buffer. We can detect 6 molecules of RT in 10 microliters of buffer containing over 1 pmol of BSA (approximately 0.1 micrograms).

Our work with RT has been very informative, but we believe that much more general approaches to exquisite detection are possible. We believe that we now have an approach which will allow us to exquisitely detect vast numbers of molecules of interest. Bringing this approach to practice is the subject of our current research. The uses of exquisite detection are manifold:
  • For the detection of toxic substances on the battlefield or at points of entry such as airports. We call this particular application of exquisite detection `the ultimate canary'.
  • For the detection of toxins or pollutants in the industrial sector.
  • For the detection of contraband.
  • For the very early detection of disease. Some diseases appear to have 'signature molecules' associated with them. These molecules are present only when the disease is present. Exquisite detection might allow for the detection of these substances in serum before significant amounts have built up and hence before the pathology has progressed.
  • For general scientific investigation.

Remark : It is important to distinguish exquisite detection from other forms of sensitive detection. Apparently, the human eye can detect single photons and the nose can detect small numbers of molecules of certain substances. Physicists can study single molecules using molecular tweezers and microscopists can see them under scanning microscopes. These are examples of the extraordinary specificity and sensitivity of detectors which work only when the target is brought to them. These methods do not allow for the detection of a single molecule from a large volume. In such a setting, the single molecule of interest has virtually no chance of ever hitting the detector and being `seen'. Exquisite detection requires the active `search' for a target molecule in a large volume. Each form of detection has applications for which it is appropriate. To put it in simple terms, seeing a needle if it is held up before us, does not mean it is easy to know if there is one in a haystack.