Common Problems with Nucleic Acid Extraction

Extracting nucleic acids from a sample isn’t easy – here are some problems that are commonly encountered

BACKGROUND

The coronavirus pandemic has transformed many aspects of our daily lives – be that how we work, how we socialise or how we buy things. This has in turn affected entire industries, with very few immune from the sweeping tides of change forced by radically different consumer behaviours. Healthcare is certainly not separate from this, with mass testing being a crucial component that enables individuals to go about their business as close to normality as possible.

With this surge in demand for coronavirus testing has come an increase of laboratories being set up to provide a supply of PCR tests. Not all of these are set-up for the high level of throughput that justifies the implementation of a fully automated, end-to-end black box solution. For smaller throughput laboratories, or high-throughput laboratories which require a secondary system, they may need to manually carry out an extraction step to extract the nucleic acids from the samples, before analysis. With that in mind, this is a guide which explains some of the common problems encountered with nucleic acid extraction.

HISTORY

Before the 1970s, nucleic acid extraction was very limited, as the methods available were only suitable for a small number of samples and resulted in small yields and low purities. A traditional method used for extraction was to carry out an exacting series of precipitation and washing procedures1 – which were both complex and time-consuming to perform. The intricacy and high number of steps meant an inevitably high level of degradation, sample loss and cross-contamination of samples.

Since then specially functionalised magnetic particles were developed to enable something called magnetic bead extraction, which was able to achieve better yields at higher purity. With an appropriate buffer system it became possible to do this quickly and efficiently.

COMMON PROBLEMS

A loss of sensitivity and false negatives can occur if magnetic beads are used without elution. This happens as the magnetic beads, their stabiliser or their metal oxides can inhibit the enzymatic detection and amplification methods further down the line. To avoid this, make sure that there is an elution step. This can be achieved by using a higher ionic strength, heating the sample or changing the ph.

The NC-15 system uses a pre-filled reagent plate which has an eluate in the first and 7th column, and also heats the sample to a liquid temperature of 65 degrees Celsius.

Manual magnetic bead extraction suffers from problems similar to the cruder traditional extraction methods, as they are labour intensive with a high susceptibility to human-error. It also requires numerous steps, of which every extra step is another chance for cross-contamination. An automated nucleic acid extraction system overcomes these obstacles by using robotics to not only remove the human-error factor, but also utilising the precision that robotics can offer to enable a higher yield that is of higher purity.  

No procedure is without the risk of error however, and there are a couple to consider with an automated system. One is to make sure that, similar to any other pipetting procedure, you change over the plastic tips that sheath the magnetic rods between runs to make sure that there is no cross-contamination. You need to make sure that these comb-style tips are inserted for every extraction procedure, as if they are not the magnetic beads will be in direct contact with the magnetic rods and therefore bind to them. This can be easily resolved however by wiping down the rods with a cloth moistened with 70% alcohol.

Also remember that any machine handling live samples should be decontaminated between runs. The NC-15 system has a built in UV light which can be switched on between cycles, for an advised time period of 12 minutes. Interior surfaces should also be wiped down with a cloth moistened with 70% alcohol. This will prevent cross-contamination.

CONCLUSION

In conclusion, laboratories can harness the power of innovation in the molecular testing area by using the magnetic bead extraction method to quickly and efficiently separate nucleic acid from the crude sample. Using an automated nucleic acid extraction system reduces or eliminates many of the risks associated with traditional manual extraction methods, and by following a few common-sense procedures you can achieve a high nucleic acid yield very simply. Using this guide should help you to navigate the difficulties encountered with extracting nucleic acids.

1                     Magnetic particles for the separation and purification of nucleic acids (nih.gov)