Evaluating the quality of genomic DNA after extraction is a critical step before initiating downstream applications such as PCR, next-generation sequencing (NGS), or genotyping. High-quality DNA ensures data accuracy, assay reliability, and optimal resource utilization.

This article outlines the five key metrics commonly used to assess the quality of extracted genomic DNA from quantification to functional validation.

Why Assess DNA Quality?

Genomic DNA extracted from biological samples can vary significantly in quantity, purity, and structural integrity. Contaminants such as proteins, RNA, salts, and residual chemicals can interfere with downstream processes, leading to suboptimal results or failed experiments. Systematic quality assessment helps confirm that DNA yield meets assay requirements, ensure DNA purity is within acceptable thresholds, verify the integrity and suitability of DNA for intended applications

1. DNA Concentration and Yield

Quantification is the first checkpoint. Reliable measurement of DNA concentration ensures sufficient input material for subsequent steps.

Common Quantification Methods:

• Fluorometric (e.g., Qubit)
  Fluorometric methods use DNA-specific dyes to bind double-stranded DNA, minimizing interference from RNA and proteins. They offer higher sensitivity and accuracy, especially for low-yield or impure samples.

• Spectrophotometric (e.g., NanoDrop)
  This method estimates DNA concentration by measuring absorbance at 260 nm. While rapid and convenient, it can overestimate DNA content in the presence of RNA or phenolic contaminants.

Recommendation: Use fluorometric methods for accurate quantification, particularly with clinical or environmental samples where yield may be low.

2. Purity Assessment Using Absorbance Ratios

Purity is assessed using absorbance ratios obtained from spectrophotometric readings:

• A260/A280 Ratio
  Indicates protein contamination. Pure DNA typically has a ratio between 1.7 and 1.9. Lower values suggest residual protein or phenol.

• A260/A230 Ratio
  Reflects the presence of salts, carbohydrates, and organic solvents. Ideal values are above 2.0. Lower ratios indicate contamination by extraction reagents or polysaccharides.

Example: DNA extracted using optimized protocols often shows ideal A260/A280 and A260/A230 ratios, indicating suitability for sensitive downstream applications like qPCR or NGS.

3. DNA Integrity and Fragmentation

Integrity refers to how intact the DNA strands are. Fragmented or degraded DNA can reduce performance in long-read sequencing, genome assembly, or multiplex PCR.

• Agarose Gel Electrophoresis
  Allows visual inspection of DNA size and integrity. High molecular weight DNA appears as a sharp, distinct band, while degradation is indicated by smearing.

• Automated Systems (e.g., TapeStation, Bioanalyzer)
  Provide size distribution, quantification, and integrity scores (e.g., DNA Integrity Number, DIN) with greater precision and reproducibility.

Note: For applications requiring high molecular weight DNA (e.g., PacBio or Oxford Nanopore), integrity assessment is critical.

4. RNA Contamination

RNA co-extraction is common and can inflate DNA concentration measurements, particularly when using spectrophotometry.

• Qubit Fluorometry minimizes this issue by using DNA-specific dyes.

• RNase treatment prior to quantification can effectively eliminate RNA contaminants.

Accurate assessment and removal of RNA is essential for workflows requiring precise DNA input amounts, such as qPCR or NGS library preparation.

5. Functional Quality: Amplifiability and Performance

In certain workflows, evaluating whether the DNA can be efficiently amplified or sequenced is the most definitive quality check.

• qPCR (quantitative PCR):
  Lower quantification cycle (Cq) values indicate good-quality, amplifiable DNA.

• Restriction enzyme digestion or ligation efficiency:
  Useful for checking compatibility with cloning or enzymatic reactions.

These assays offer an added layer of confidence, particularly for clinical, diagnostic, or regulatory applications.

The quality standards and requirements for genomic DNA can vary significantly depending on the intended downstream application and the type of starting material. For instance, DNA extracted from whole blood is typically expected to yield high-molecular-weight DNA with A260/A280 ratios around 1.8 and minimal RNA contamination — suitable for applications like NGS or genotyping. In contrast, DNA extracted from complex matrices like stool or soil often requires more rigorous purification and integrity checks due to the presence of inhibitors and microbial DNA.

What sample types do you work with?

Are you navigating the QC challenges of low-yield or inhibitor-rich samples? Whether you're working with blood, buccal swabs, FFPE, or environmental samples, each type presents unique quality control hurdles.

Explore Manta's QC benchmarks across sample types
See how Manta performs across diverse sample types and QC thresholds here.