Biología de síntesis
Miniaturización y aceleración del ensamblaje de ADN y CC de la síntesis de genes
The applications of DNA sequencing in biological research are growing. A significant driver of this growth is next-generation sequencing (NGS), a modern DNA sequencing technology instrumental in achieving complete DNA sequences or genomes of humans, many animals, plants, and microbial species.
NGS can offer rapidity, reliability, and cost-effectiveness. However, these benefits also depend on the quality of the DNA being sequenced. Poor quality or inefficiency in the early stages of the sequencing process can mean additional costs of errors, rework, or waste later. When DNA sequencing drives targeted drug development, testing, and manufacturing, or the tracking and containment of dangerous viruses and epidemics, outcomes must not be put at risk through ill-advised shortcuts upstream.
Cleanup kits are a pivotal part of the NGS process. They have an immediate impact on efficiency and on quality, as well as key impacts downstream. Clearly, it is not worth jeopardizing entire DNA sequencing operations and more, simply to save pennies on the price per sample of cheaper but lower performance, poorer quality, and less efficient kits.
It is therefore critical to examine a DNA sequencing cleanup kit before selection and compare its overall performance with others, either when starting NGS operations or when considering changing to a different cleanup kit solution. While cleanup kit cost is always a factor, switching to cheaper or other products is not necessarily more advantageous and focusing exclusively on price can have highly undesirable consequences.
Factor 1 – Yield
The first and perhaps most obvious factor is percent yield, the final DNA concentration divided by the starting DNA concentration, multiplied by 100%. For optimal quality and efficiency, the percent yield should be between 80% to 95%. Lower yields may compromise downstream data quality. Not all cleanup kits can achieve consistently high yield (see Figure 1).
Factor 2 – Purity
Good quality DNA should have an A260/A280 ratio of at least 1.8. A lower rating indicates contaminating proteins. Similarly, an A260/A230 ratio of less than 2.0 indicates other contaminates.
Factor 3 – Time
Time is often the most valuable resource. Shorter cleanup times are better. A difference between 30 and 45 minutes per cleanup step (see figure 2) is multiplied each time an additional cleanup step is needed when running library construction kits. For example, 3 cleanup steps mean a difference between 1.5 hours and 2.25 hours. This difference is multiplied again as overall NGS activity increases.
Factor 4 – Predictable Performance
Uniformity of DNA fragment size affects both quality and efficiency of DNA sequencing. Initial fragment size must be correctly targeted to allow for additional base pair (bp) length as adapters are added to fragments for sequencing. Ideally, fragment size concentrations will remain high and predictable as different binding ratios are used (see graph for AMPure XP in Figure 3). This is an important factor if a user is considering switching from one cleanup kit to another. Traces below show the relative differences in peak height and recovery (Figure 3).
Factor 5 – Risks of Switching
The example above of binding ratios giving different DNA fragment size concentrations for different cleanup kits is one risk of switching to another cleanup kit. Others are:
- The extra time and effort needed to evaluate a new cleanup kit
- The risk of having to redo sequencing later if the new product does not perform as well as the original product
- A library kit supplier withdrawing its support for resolving issues if used “off-label” with a cleanup product that was not recommended by that library kit supplier
Cheap DNA sequencing cleanup kits may perform poorly and jeopardize the overall results of NGS. Risks include wasted time, sub-optimal efficiency, and incomplete discovery.
By comparison, AMPure XP targets what really matters to businesses and organizations working with DNA sequencing: consistency and reliability of performance lot-to-lot, quality for manufacturing, and on-time delivery.
AMPure XP is recommended by 215 library construction kit manufacturers. The suppliers of these kits recommend AMPure XP often as the sole cleanup option. AMPure XP has also been cited in over 21,000 articles on Google Scholar.
Using AMPure XP as your cleanup kit, you can keep your sequencing projects on track and continue to provide the best results possible to your customers, patients and journals.
Beckman Coulter makes no warranties of any kind whatsoever express or implied with respect to these protocols, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringement. All warranties are expressly disclaimed.
Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in diagnosis of disease or other conditions. These protocols are for demonstration only and are not validated by Beckman Coulter.
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