Genomic DNA

Understanding genomic DNA in both sequence and structure, furthers the understanding of evolution, the species concept, genetic influences on disease, genetic diseases, and so much more.


What is genomic DNA?

First isolated in the mid-1800s, genomic DNA, commonly referred to as gDNA, comprises the genome in nearly all living organisms. Complex in sequence and structure, genomic DNA provides significant challenges to researchers. In fact, scientists have spent entire careers furthering our understanding of the sequence and structure of genomic DNA. The sequence of genomic DNA carries information in a quaternary code organized in genes and other discrete genetic elements. Access to this information is regulated by chemical and physical modifications to the genome, which is to say the binding of small molecules and proteins to the genomic DNA, and the physical and spatial organization of the genome.

Downstream applications including PCR, NGS, and microarrays, among others, require the use of gDNA that meets applicable standards. Traditional methods of genomic DNA quality control analysis are limited in accuracy and precision, potentially compromising downstream analysis. Agilent - formerly Advanced Analytical (AATI) manufactures several instruments and reagent kits well suited to the accurate quantification, qualification, and sizing of genomic DNA.

The accurate assessment of genomic DNA size, quantity, and quality is crucial to downstream applications including: PCR, genotyping, and next-generation sequencing. gDNA – whether intact, FFPE, or from ancient samples – provides scientists with insight into the underlying biology of cancer and other diseases, expands the species concept, elucidates evolutionary relationships between organisms and within populations, and so much more. All of these applications start with ensuring the use of high quality gDNA, read on to learn more about the superior gDNA analysis provided by Agilent - formerly AATI.

Problems with gDNA Analysis

Genomic DNA provides researchers a rich source of information for understanding everything from the most fundamental aspects of evolution to developing novel cancer treatments. For researchers to take advantage of this resource, high-quality gDNA must be used for sophisticated analysis methodologies such as next-generation sequencing (NGS). Legacy methods of gDNA quality control analysis – such as agarose gel electrophoresis – demand high concentrations of sample for successful analysis and lack the resolution to accurately and reliably size intact gDNA.

Intact Genomic DNA

Genomic DNA provides several analytic challenges, namely the size and high or low concentrations of sample. Most methods used for genomic DNA analysis, such as agarose gel electrophoresis, demand a high input concentration and lack the necessary resolution and sizing capacity to reliably and accurately assess gDNA size, quantity, and quality. The Fragment Analyzer, 5400 Fragment Analyzer, and the Femto Pulse System provide superior assessment of intact genomic DNA for various applications, from PCR and genotyping to next-generation sequencing.

Figure 1. Genomic DNA separated using the Genomic DNA 50kb Kit (DNF-467) on a Fragment Analyzer equipped with a Short Capillary Array (33-55). The GQN was applied to this sample with a Size Threshold of 10,000 bp and a calculated GQN of 9.0.

The Fragment Analyzer and the 5400 Fragment Analyzer provide researchers with robust gDNA analysis solutions to fit any throughput need, from 12 samples in about one hour, to over 2,400 samples in 24 hours. The Genomic DNA 50kb Kit (DNF-467) provides confident assessment of gDNA smears through 60 Kb. A single 200-fold dilution greatly simplifies sample preparation, conserving researcher time. Similarly, the HS Genomic DNA 50kb Kit (DNF-468) offers sizing of gDNA smears through 60 Kb, though without a 200-fold dilution.

The empirical assessment of gDNA quality is assisted by the Genomic Quality Number (GQN), a dynamic quality metric designed by Agilent - formerly AATI for ProSize Data Analysis Software. A Size Threshold is selected by the researcher, and the GQN for the gDNA smear is reported as a value between 0 and 10, with higher values corresponding to more of the sample meeting the user-defined standards.

Figure 2. Smear Analysis of a genomic DNA sample. The Smear Range – designated by the dotted red lines – covered from 1,500 bp – 129,477 bp. Smear Analysis determined an average smear size of 59,296 bp, shown in red numbers on the electropherogram and the inset table. The Genomic DNA 50kb Kit (DNF-467) was employed for this separation. Capillary electrophoresis was performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55).

The Smear Analysis function in ProSize provides smear specific analysis of concentration and size (bp) of gDNA smears, providing researchers with the accuracy and precision demanded by downstream applications.

Figure 3. Large genomic DNA smear separated with the Genomic DNA 165kb Kit (FP-1002). Pulsed-field capillary electrophoresis was performed using a Femto Pulse System equipped with a Short Capillary Array (22-47). On the electropherogram: the dotted blue line represents the Size Threshold selected for GQN determination, the vertical red lines denote the Smear Analysis Range (1,000 bp – 393,664 bp). The GQN was determined to be 8.6 with a Size Threshold of 50,000 bp.

For large genomic samples over 60 Kb, the Genomic DNA 165kb Kit (FP-1002) for the Femto Pulse System is the ideal solution. Using pulsed-field capillary electrophoresis, this gDNA smear analysis solution is capable of sizing smears through and unprecedented 165 Kb. Furthermore, the ProSize analysis tools – GQN and Smear Analysis – work flawlessly with the large gDNA smears analyzed with the Genomic DNA 165kb Kit (FP-1002).

Degraded and FFPE Genomic DNA

In addition to the standard analytic challenges posed by genomic DNA, formalin-fixed, paraffin-embedded (FFPE) gDNA and gDNA that has been otherwise degraded present unique challenges for QC analysis. Notably, gDNA extracted from FFPE tissue often undergoes shearing and crosslinking with proteins and other DNA molecules. Such degradation frequently leads to a complex and convoluted separation profile. The Fragment Analyzer, the 5400 Fragment Analyzer, and the Femto Pulse System provide the means and tools to address the QC challenges of FFPE gDNA head on.

Quality scoring of FFPE gDNA and degraded gDNA is complex, often requiring researchers to guess. The GQN calculated in ProSize Data Analysis Software provides researchers with an empirical quality scoring strategy. Users simply have to select a Sizing Threshold deemed appropriate for the sample(s) of interest and proceed from there, quickly identifying samples that meet their unique requirements.

Figure 4. FFPE genomic DNA separated using the Genomic DNA 50kb Kit (DNF-467). Capillary electrophoresis was performed with a Fragment Analyzer equipped with a Short Capillary Array (33-55). A Size Threshold of 1,000 bp – denoted by the dotted blue line – provided a GQN of 3.7.

Cell-Free DNA

Cell-free DNA (cfDNA) offers researchers and clinicians a diagnostic gold mine. Generated by the cleavage of genomic DNA between nucleosomes, cfDNA is produced by healthy, diseased, and dying cells. Commonly isolated from plasma and serum, scientists are isolating cfDNA from other fluids such as urine and saliva, increasing its diagnostic utility.

Studied via NGS, cfDNA must meet rigorous quality standards to ensure successful sequencing. Legacy methods of nucleic acid QC analysis lack the sensitivity and resolution to reliably assess cfDNA. Learn more about how the instruments from Agilent - formerly AATI provide superior analysis of cfDNA.

gDNA / Genomic DNA Analysis Kits

Agilent - formerly AATI offers several instrument-based solutions for the effective and reliable QC analysis of gDNA: the Fragment Analyzer, the 5400 Fragment Analyzer, and the Femto Pulse System. These instruments, in combination with the appropriate reagent kits, provide superior analysis of genomic DNA with unmatched resolution and sizing capabilities.

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