First isolated in the mid-1800s, genomic DNA (gDNA) comprises the genome in nearly all living organisms. The genome contains all an organism’s biological information to be passed on to the next generation. By studying genomics, scientists can gain a deeper understanding about the functions, interactions, and regulation of specific genes that control the growth and development of an organism. Complex in sequence and structure, gDNA provides significant challenges to researchers. The sequence of gDNA carries information in a quaternary code organized in genes and other discrete genetic elements. Access to this information is regulated by the genome’s organization and chemical and physical modifications to the genome, including binding of small molecules and proteins to the gDNA.
Genomic DNA Quality Assessment
Most gDNA QC analysis methods, such as agarose gel electrophoresis, demand a high input concentration and lack the necessary resolution and sizing capacity to reliably and accurately assess gDNA integrity. The Agilent Automated Electrophoresis instruments offer different advantages in terms of throughput, sensitivity, speed, and resolution.
In addition to the standard analytic challenges posed by genomic DNA, formalin-fixed, paraffin-embedded (FFPE) gDNA and gDNA that has been otherwise fragmented or degraded present unique challenges for QC analysis. The Agilent Automated Electrophoresis system provide the means and tools to address the QC challenges of FFPE gDNA.
Several instrument offerings from Agilent provide effective solutions for the QC analysis of DNA and RNA samples. With the relevant reagent kits, the instruments provide unmatched sizing and quantification capabilities.
Downstream applications for gDNA include PCR, next-generation sequencing (NGS), microarrays, gene editing, cloning, and genotyping, among others. These applications require the use of gDNA that meets applicable standards. This assessment can be difficult as gDNA provides several analytical challenges for size, quality, and concentration. Most gDNA quality control (QC)analysis methods, such as agarose gel electrophoresis, demand a high input concentration and lack the necessary resolution and sizing capacity to reliably and accurately assess gDNA integrity. The Agilent Automated Electrophoresis portfolio is now comprised of several instruments, including the TapeStation and Femto Pulse systems, each offering different advantages in terms of throughput, sensitivity, speed, and resolution. Together, the Automated Electrophoresis instruments provides superior assessment of intact gDNA, including accurate qualification, sizing, and quantification.
Fragment Analyzer systems
The Fragment Analyzer systems 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. There are two kits specifically designed to analyze gDNA:
The Genomic DNA 50 kb kit (p/n DNF-467) provides confident assessment of gDNA smears through 60 kb. A single 200-fold dilution greatly simplifies sample preparation, conserving researcher time.
The HS Genomic DNA 50 kb kit (p/n DNF-468) offers sizing of gDNA smears through 60 kb, for lower concentration samples.
The Genomic Quality Number (GQN), a dynamic quality metric reported by ProSize data analysis software, is available with both kits and provides an empirical assessment of gDNA quality. A size threshold is selected by the researcher, and the GQN for the gDNA smear is reported as a value between 0 and 10. A higher GQN value corresponding to more of the sample meeting the user-defined standards.
Genomic DNA separated using the Genomic DNA 50 kb kit on a Fragment Analyzer system equipped with a short capillary array. The GQN was applied to this sample with a size threshold of 10,000 bp (purple line) and a calculated GQN of 9.0, indicating high-quality gDNA.
The Smear Analysis function provides specific analysis of concentration and size (bp) of gDNA smears within a user-defined sizing range, providing researchers with the accuracy and precision demanded by downstream applications.
Smear Analysis of a gDNA sample analyzed on a Fragment Analyzer system using the Genomic DNA 50 kb kit with a short capillary array. The smear range can be visualized by the dotted red lines. The size of the highest peak within the smear is indicated by the blue number over the peak. In contrast, the Smear Analysis determines an average smear size, shown by the red number on the top of the electropherogram and in the inset table.
The TapeStation systems offer easy and fast automated electrophoretic separations of genomic DNA.
The Genomic DNA ScreenTape assay (p/n 5067-5365 and 5067-5366) provides accurate quantitation and reliable quality analysis of up to 96 samples from 200 through 60,000 bp in less than two minutes per sample.
The use of gDNA for downstream applications, such as NGS, relies on having high-quality input samples. The TapeStation software provides an automated and objective quality score, the DNA integrity number (DIN). The DIN ranges from 1 to 10 and indicates the quality of a gDNA sample by assessing the distribution of the sample across the entire sizing range. A lower DIN correlates to more degraded sample, while a high DIN would indicate a good quality sample, allowing you to make informed decisions for your downstream applications.
gDNA samples at four degradation stages were analyzed on a TapeStation system with the Genomic DNA ScreenTape assay to determine DNA integrity (DIN) and sample concentration. A) Gel image with corresponding DIN displayed below each lane. B) The corresponding electropherogram overlay of all 12 gDNA samples.
Femto Pulse system
For applications that require more detailed information and accurate sizing above 60 kb, such as long read sequencing, the Femto Pulse system provides the ideal solution:
The Genomic DNA 165 kb kit (p/n FP-1002) uses pulsed-field capillary electrophoresis, to size high molecular weight gDNA smears through an unprecedented 165 kb, in about 1.5 hours.
Additionally, the ProSize data analysis software tools used with the Fragment Analyzer systems, such as the GQN and Smear Analysis function, work flawlessly with the large gDNA smears analyzed on the Femto Pulse system, providing you even more detailed information about your high molecular weight samples.
Large gDNA smear separated with the Genomic DNA 165 kb kit. Pulsed-field capillary electrophoresis was performed using a Femto Pulse system equipped with a Femto Pulse 12-Capillary Array. On the electropherogram, the dotted blue line represents the size threshold selected for GQN determination, and the vertical red lines denote the Smear Analysis range. For this sample, with a user-defined threshold of 50,000 bp, the GQN is 8.6, indicating that 86% of the sample is larger than the user-defined threshold.
For Research Use Only. Not for use in diagnostic procedures.
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 fragmented or degraded present unique challenges for QC analysis. Notably, gDNA extracted from FFPE tissue often undergoes shearing and cross-linking with proteins and other molecules. Such degradation frequently leads to a complex and convoluted separation profile. The Agilent Automated Electrophoresis systems, including the Fragment Analyzer, TapeStation, and Femto Pulse systems, provide the means and tools to address the QC challenges of FFPE gDNA.
Quality scoring of FFPE gDNA and degraded gDNA is complex, often requiring researchers to make a best-guess when using conventional agarose gel electrophoresis.
The Fragment Analyzer and Femto Pulse systems use ProSize data analysis software to calculate a GQN, providing researchers with an empirical quality scoring strategy. Users simply define a sizing threshold deemed appropriate for the samples of interest and can then quickly identify samples that meet their unique requirements.
The Genomic DNA ScreenTape assay for the TapeStation systems provides an objective measurement of DNA integrity through the DNA integrity number (DIN).
While FFPE gDNA generally has a lower integrity, knowing the quality of the sample can allow a user to modify certain workflows for these samples. For example, fragmentation conditions could be adapted based on a sample’s initial integrity. Get the knowledge you need about your samples with the Agilent Automated Electrophoresis portfolio of instruments.
Fragment Analyzer system
FFPE gDNA separated using the Genomic DNA 50 kb kit on a Fragment Analyzer system equipped with a short capillary array. A size threshold of 1,000 bp, denoted by the dotted blue line, provided a GQN of 3.7.
FFPE gDNA samples of varying integrity separated on a TapeStation system using the Genomic DNA ScreenTape assay. The results are shown as both an electropherogram overlay and a digital gel image, with the DIN listed below each gel lane.
For Research Use Only. Not for use in diagnostic procedures.
Genomic DNA (gDNA) Analysis Kits
Agilent offers several instrument-based solutions for the effective and reliable gDNA QClQC analysis of gDNA: the Fragment Analyzer, TapeStation, and Femto Pulse systems. These instruments, in combination with the appropriate assays and reagent kits, provide superior analysis of gDNA with unmatched resolution and sizing capabilities.