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Tutorials : Sep 1, 2008 ( )
Automated Purification for Genetic Trait Maps
Achieving a High Level of Purity Is Often a Challenge to the Researcher!--h2>
The single most important prerequisite for genomic research is clean starting material. Without this initial clarity, the integrity of the results can be seriously impacted, especially when looking for small differences between SNPs, for example. Achieving a high level of purity is not always easy and can take up a lot of a researcher’s time, a process that is often compounded by the need to process hundreds of samples at once. Manual methods add to the problem of sample consistency, both within and between experiments.
In this tutorial, we look at how automated nucleic acid extraction and purification using GeneMole®, a magnetic purification system developed by Mole Genetics, has enabled scientists to overcome problems related to genetic trait mapping.
A Dog's Tale
A team of scientists, lead by Professor Leif Andersson of the functional genomics group in the department of medical biochemistry and microbiology at Uppsala University, was able to prove, using genetic material from dogs, that it is possible to map traits efficiently using a two-stage strategy. In the first stage, genome-wide mapping with a relatively sparse marker set (~15,000 SNPs) in a single breed, using as few as 10 “affected” and 10 control animals, identified a ~1 Mb region of association.
In the second stage, the region of association was narrowed to a few hundred kilobases by performing fine mapping with a dense set of SNPs across multiple breeds. Full sequencing of this narrow region then enables the genes and related mutations for certain phenotypic traits to be proposed.
Manual DNA preparations, which are the basis of such work, are notoriously laborious and time consuming. In projects that rely on a large amount of high-quality DNA, this is not only rate-limiting but also introduces a significant amount of unavoidable human error.
Automating the standard chemical or column methods, however, is difficult since they involve multiple steps, many of which require centrifugation or other condensation/concentration processes.
The advent of superparamagnetic particles with controllable surface properties has enabled a different system for preparing DNA (as well as proteins, cells, and cell components). An overview of the simplicity of isolating DNA from a generic source using GeneMole is shown in Figure 1. In outline: DNA is bound to the magnetic beads and can be held by a magnet so that the buffers can be added and removed without moving the sample. This significantly reduces reagent carryover and also increases yield.
In addition, all the required reagents are prefilled in sealed and disposable strips to secure minimal interference with the precious sample. Mole Genetics’ One Strip-One Sample™ principle offers flexibility with the choice of 1 to 16 samples per single run. As a result, this process produces high quantities of pure DNA whatever the throughput requirements.
Undock and Load
Once the instrument is switched on, users are prompted to select the required program, and then specify the sample and elution volume to be used. The worktray is then taken from GeneMole and prepared—the correct number of barrier tips, microtubes for elution, and waste containers are loaded and then the samples (in 1.2 mL microtubes) are loaded.
Once this worktray is placed back in GeneMole and the door closed, the selected protocol can be started using the start button on the touchscreen.
Ease of Use
GeneMole performs nucleic acid extraction based on the use of paramagnetic beads. The method involves lysis of cells and binding of the DNA to paramagnetic beads. Impurities are removed in two washing steps, before highly purified DNA is finally eluted off the particles in a chosen elution volume.
In the lysis step, lysis buffer is added to the samples to release the DNA. Magnetic beads are then added to bind to the free DNA.
In the wash step, a magnet is applied to the outside of the tubes and the beads (plus DNA) are held against the inside edge while the lysate is removed and the first wash buffer added. The beads are then released by removing the magnet to allow the DNA to be washed. The magnet is reapplied, and the wash procedure repeated to ensure all lysate and impurities are removed.
In the elution stage, the correct volume of elution buffer is added to the beads/DNA and the DNA is resuspended. The magnet is reapplied to hold the now unbound magnetic beads against the tube. This means that the elution buffer plus DNA can be pipetted into clean tubes without any beads.
The performance of GeneMole has been assessed based on yield, purity, cross-contamination, integrity, and reproducibility. Results are based on human blood samples as source material.
Whatever the sample, it is important to ensure that there is good yield and purity, and that these are consistent (Figure 2).
A checkerboard setup of DNA extraction was designed to validate the absence of cross-contamination during the extraction procedure. Runs of 16 samples were performed, where human blood was substituted with dH2O in every other sample. Results showed no evidence of cross-contamination.
DNA extraction on the GeneMole showed good reproducibility when tested on 16 aliquots of a human blood sample (Figure 3).
Human blood samples stored in common anticoagulants—EDTA, heparin and citrate—were processed on GeneMole and found to have similar results for yield and quality.
Professor Andersson’s group processes several thousand samples per year from many different domestic species. As a result of using GeneMole automated magnetic bead processor the group has reported an increase in throughput. Each of the different sources they process—dog, sheep, cattle, horse, chicken, and pig—has slightly different properties but the GeneMole system produces consistent results across all species, even for chicken blood where the nucleated erythrocytes can cause spin columns to clog and therefore reduce yield.
What has also become clear from their research, according to Prof. Andersson, is that the GeneMole system provides good DNA integrity with fewer DNA fragments and an eluate that is protein free. This is essential for the sensitive downstream processes involved in genetic trait analysis.
With a large research group, GeneMole also ensures consistency within a sample set and also across sample sets processed by different researchers. This makes one set of data more comparable to another set than would be the case with manual pipetting. This is a great advantage since data sets can be produced over a number of years and it is essential that the first set is processed in exactly the same way as the last.
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