Start With Isolating DNA
All genomic analyses start with isolating DNA. This DNA could be genomic DNA from intact organisms, frozen tissues, or from fixed tissues embedded in paraffin (FFPE). It may include PCR amplicons that are used as intermediate steps in a cloning or sequencing reaction. Finally, the construction of next generation sequencing libraries involves the manipulation of pools of DNA fragments.
Analyze the Amount and Integrity of the Isolated DNA
In all of these procedures, investigators need to analyze the amount and integrity of the DNA isolated before moving on to more complicated or expensive steps. This ensures efficient use of budget, unbiased NGS results, and general data reliability and reproducibility for all downstream applications.
- DNA extracted from FFPE-preserved tissue to examine the level of DNA fragmentation that limit the usefulness of information from subsequent assays;
- NGS library QC to determine the size distribution and abundance of library fragments to predict NGS success;
- Success of PCR amplification prior further manipulation.
Agarose Gels for Analyzing DNA Fragments
Agarose gels have long been a standard for analyzing DNA fragments, but while they are inexpensive, they are not very sensitive, precise or high throughput. Other platforms have been developed that provide better sensitivity and higher throughput. However, these often rely on expensive specialty chips or reagents. In addition, each of these solutions require a dedicated instrumentation, taking up more space in the lab and requiring additional training and maintenance costs.
The Need for Reliable, Cost-effective dsDNA Integrity Testing
Putting Your CE-based Genetic Analyzer to Work
Scientists at Thermo Fisher Scientific developed a method for double-stranded DNA (dsDNA) fragment QC that runs on Applied Biosystems™ existing capillary electrophoresis (CE) genetic analyzers. Fragment analysis by CE yields quantitative information, while providing further library information such as the size of DNA fragments. This method does not require the purchase of specialty polymers or capillaries, therefore providing additional flexibility and utility for the existing CE systems including the 4-cap SeqStudio Genetic Analyzer and the latest mid-throughput SeqStudio Flex Series Genetic Analyzers.
A New Twist on Fragment Analysis
Turning Dyes and DNA Digests into Data
The new protocol relies on incubating DNA with a double-stranded DNA intercalating dye, followed by electrophoresis using existing polymers and buffers. One key difference between this dsDNA QC protocol and standard fragment analysis is performing CE separation at low temperature. This maintains the DNA in a partial duplex with the intercalating dye that can be detected as a positive signal. The resulting electropherogram can be examined for relative size(s) of a sample and provide a semi-quantitative measurement of the amount of DNA in the sample.
Details of the method are provided in protocol entitled Analysis of double-stranded DNA fragments using capillary electrophoresis.1 Briefly, TOTO™-1, an inexpensive fluorescent DNA intercalating dye, is incubated with a DNA sample for approximately 10 minutes in water. The sample is then run through POP-7™ polymer on 3500 or SeqStudio Flex systems, or POP-1™ on SeqStudio, with the capillary heater turned off or minimized. Concurrently, a single or set of test-defined fragments, for example phage lambda DNA digested with HindIII or phage phiX174 digested with BsuRI are run in a separate capillary. The peaks generated during electrophoresis are detected using the E5 (DS-02) filter set.
Confidently Perform NGS
Confirming Library Quality to Ensure NGS Data Quality
Once the electropherograms from samples are obtained, the size of the resulting fragment(s) can be estimated by comparing peaks to the fragment standard with known sizes. Small PCR artifacts, intact gDNA, and the fragment sizes of interest are quickly and easily visualized.
Let LIZ Standards Show You the Way
Internal Standard for Increased Precision
For additional precision, each sample can be run with a LIZ™1200 single-stranded standard. By calibrating the known double-stranded fragments to the LIZ1200 peaks, a precise determination of the size of the unknown peak can be determined. Finally, the peak height will be correlated with the amount of DNA in that peak, leading to a semi-quantitative estimate of the amount of DNA present. Of course, there are many variables that determine how much DNA is ultimately injected into the capillary. Therefore, precise quantification requires more extensive calibration and normalization using defined amounts of a similar-sized fragment.
In summary, this method provides a path to fast and simple QC of dsDNA samples using existing Applied Biosystems genetic analyzers.
- Analysis of double-stranded DNA fragments using capillary electrophoresis. Thermo Fisher Scientific Application note.