As traditional molecular biology techniques such as PCR and microarray analysis continue to become standard methods for molecular diagnosis, quality control steps that minimise the loss of clinical samples are essential. A novel method of quantifying biomolecules using only 1µL of sample is ideal for diagnostic workflows.
by Dr P. Desjardins
The microsample quantitation method was first introduced to the scientific community in 2000 by NanoDrop Technologies (now part of Thermo Fisher Scientific). The fundamental concept removes traditional containment devices such as cuvettes and capillaries. The patented sample retention system combines fibre optic technology and the natural liquid surface tension to capture and hold a 1µL sample between two optical surfaces during measurement. By eliminating traditional containment devices, the volume of the required sample is greatly reduced thus conserving most of the sample for downstream analysis. The NanoDrop 1000 Spectrophotometer was the first instrument developed to use the invention for microsample analysis. The user simply loads 1µL of sample onto a lower optical surface. An upper optical surface engages the sample to create a liquid column [Figure 1].
The distance between the optical surfaces is precisely controlled and forms the path length during the absorbance measurement. The absence of traditional containment devices allows the path length to change in real time. This provides for an exceptionally large dynamic range of potential concentrations to be measured. As the path length is shortened, higher concentrations of sample can be measured. (i.e. 2 ng/µL to 3700 ng/µL for dsDNA), essentially eliminating the need to perform dilutions.
To address the demands of higher throughput environments, the NanoDrop 8000 spectrophotometer was design to perform up to eight 1µL measurements
simultaneously [Figure 2].
Although NanoDrop instruments have been widely accepted in common research environments, they are increasingly used for clinical applications. The differentiating factor between molecular biology techniques performed in basic research environments and the same techniques performed in medical settings is that the results are for clinical use. Due to the frequently limited amounts of material in clinical samples, reducing the volume required for quality control steps is important, and is the main reason that the NanoDrop microsample quantitation method is being adopted in several areas of molecular diagnostics.
One example is for sequence-based genotyping, where microsample technology is being used to quantify critical biomolecules at several steps during the diagnostic workflow. After a clinical specimen is acquired, DNA extraction is performed. Using a minute amount of elution, the NanoDrop 1000 determines the concentration and purity of the extracted sample. This information is critical for optimising the next step in the process: DNA amplification by PCR (polymerase chain reaction). The 1µL quality control measurement not only conserves the maximum amount of the original genetic material, it allows the clinician to determine the smallest amount of template DNA that can be used for a successful PCR reaction. Post amplification, the instrument can also be used to measure the final concentration of PCR product. This measurement is used to optimise the sequencing reaction, which requires a specific ratio of DNA to primer concentration. By using microsample quantitation instrumentation, quality control steps are easy to perform throughout the process, without compromising accuracy or consuming large portions of precious sample. The same is true for many molecular diagnostic workflows, such as microarray-based diagnostics and tissue typing for patient-donor crossmatching.
Furthermore, laboratories involved in medical research are continually developing new clinical tests that use molecular biology techniques. For example, the development of solid tumour testing is often extremely difficult due to the small amounts of available tumour cell mass. The samples are often difficult or simply impossible to reobtain, and the amount of genetic material extracted from a specific solid tumour may be so limited that the only possible method of measuring the sample is by microsample quantitation.
As more molecular biology techniques are integrated into the clinical setting, microvolume quality control steps that are successful in the research environment can be applied to molecular diagnostics. By consuming minimal genetic material derived from precious medical samples, NanoDrop technology is proving to be an important tool for quantifying biomolecules at key steps throughout clinical
diagnostic workflows.
The author
Philippe Desjardins
Scientific Marketing Manager NanoDrop instruments
Thermo Fisher Scientific.
Epsom, Surrey, UK
Medica Stand 1/E11
