Ensure DNA recovery and quality

This study demonstrates that research labs can achieve a substantial consumable cost savings in sample preparation without sacrificing DNA yield, purity, or quality

 

Challenges

 

DNA binding plates are employed as a basis for automated DNA isolation from blood for a broad variety of diagnostic tests. Such assays are used for disease detection, prognosis, and monitoring. Although these tests often utilize the same well-established automation platforms as basic research, they require a higher degree of robustness and sample consistency in terms of DNA yield, concentration, and quality.

 

Solution

 

There are several product options available in the market containing DNA binding plates and a complete set of buffers. Although vacuum filtration is more user-friendly and easily automated, centrifugation is still the primary method used to ensure DNA recovery and quality. An alternative to pre-packaged kits is the use of individual components, such as Pall AcroPrep Advance DNA binding plates which utilize standard glass fiber technology for genomic DNA isolation and purification. This platform offers additional flexibility as it can be used with a variety of reagent kits and, therefore, customized to your application requirements. Additionally, these plates offer a consistent and reproducible isolation of high yield and good quality gDNA from human blood using either vacuum or centrifugation.

 

Samples of fresh blood (EDTA) produced the best gDNA yield at 3.7 µg/well (100 µL blood). That represents up to 86% DNA recovery calculated based on WBC concentration in the starting blood sample. Inter-plate performance for DNA yield was similar for all nine plates used in the study varying in the range of one SD between 79-86%. Intra-plate (well-to-well) consistency evaluated by CV (%) was 11% or less. The AcroPrep Advance plates demonstrated equal to, or better, performance than the competitor’s plates when used with the Qiagen gDNA buffer kit.

 

DNA can help diagnose or indicate a susceptibility to having or passing on a wide range of diseases, such as cystic fibrosis, Huntington's disease, sickle cell anemia, and Tay-Sachs. Fresh or frozen blood is the most common specimen used for this type of analysis, and sample preparation is considered one of the more critical factors in achieving the levels of sensitivity and specificity required. Fast and efficient DNA isolation from whole blood is essential to obtain the necessary high quality starting material.

 

 

Many research labs achieve high levels of DNA sample yield, concentration, and quality while obtaining fast and efficient sample preparation using complete kits that include binding plates and buffers. Pall offers an alternative to this approach by selling DNA binding plates as a separate component that can be used with the solution kits of many manufacturers.

 

A key advantage is that the total cost of the Pall DNA binding plates and buffers can be 50% of the cost of the complete kits that incorporate binding plates and buffers. Additionally, such an approach offers added flexibility for your specific requirements, and the versatility to purify samples using either vacuum or centrifugation. The purpose of this study is to compare the DNA recovery and purity of samples prepared with the AcroPrep Advance DNA binding plate and popular commercial buffers to samples prepared with a competitor’s plate. The two different plates were used with the same materials and methods based on vacuum filtration and using a liquid-handling robotic system popular in research facilities. The results show that DNA recovery is similar for the Pall plates and the competitor’s plates.

 

The six Pall plates ranged from 71-86% DNA recovery, while the three competitor’s plates ranged from 73-79% recovery. Intra-plate (well-to-well) consistency was also similar for all nine plates with CV (%) of 11% for all nine plates, indicating that the purification process is highly reproducible. Inter-plate CV (%) was also similar for all nine plates with intra-plate consistency CV (%) of 6% or less. DNA purity is also at similar high levels for both Pall’s and the competitor’s plates. The OD260/280 ratio was in the range of between 1.73-1.83 for the six Pall plates and was between 1.72-1.79 for the three competitor’s plates.

 

The inter-plate consistency for DNA purity was in the range of one SD for all nine plates in the study with intra-plate consistency CV (%) within 6% or less. Gel electrophoresis images indicate similar levels of quality and DNA concentration on the samples produced with both the Pall binding plates and the competitor’s plates. The study also clearly demonstrates the advantages of using fresh blood samples in research studies. Fresh blood produced the highest yields at 3.7 μg/well. Blood lysate that had been stored for 5 hours produced a lower DNA yield of 2.7 μg/well. Blood that had been frozen and thawed provided an even lower DNA yield of 2.3 μg/well.

 

These results demonstrate that AcroPrep Advance DNA binding plates can be used in conjunction with commercially available buffers to deliver equal or better performance than a competitor’s plate. The combination of the AcroPrep Advance DNA binding plate and commercial buffers provides a 50% cost savings compared to the purchase of a complete kit.

 

This study demonstrates that research labs can achieve a substantial consumable cost savings in sample preparation without sacrificing DNA yield, purity, or quality

 

Results

 

AcroPrep Advance DNA binding plates provide an equal, or better, performance than competitor’s plates when used with commercially available buffers. Additionally, companies have experienced dramatic savings by implementing our binding plates. AcroPrep Advance DNA binding plates plus buffers cost about 50% less than competitor’s complete kit including binding plates and buffers. But the up-front savings are only just the beginning.

 

DNA yield and purity of Pall’s DNA binding plates is equal to or better than competitor’s plates. Inter-plate and intra-plate consistency for both DNA yield and purity of the samples produced with the Pall plates is equal to or better than the competitor’s plates. Gel electrophoresis images indicate high quality and similar DNA concentration for both Pall and competitor plates. And best of all, fresh blood provides the highest DNA yields while blood lysate stored for 5-hours and freeze/thawed blood provide substantially lower yields. For more innovative solutions from Pall’s Laboratory team, contact us today.

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Pall’s centrifugal devices simplify many common nucleic acid and protein sample preparation procedures. These devices provide efficient concentration and salt removal of samples from 50 µL to 60 mL in just minutes. Choose from membranes that have been developed to assure low non-specific biomolecule binding and typically provide > 90% recovery of target biomolecules.
Pall’s centrifugal devices simplify many common nucleic acid and protein sample preparation procedures. These devices provide efficient concentration and salt removal of samples from 50 µL to 60 mL in just minutes. Choose from membranes that have been developed to assure low non-specific biomolecule binding and typically provide > 90% recovery of target biomolecules.
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