DNA Quantification and Common Methods

DNA quantification is a routine task in regular molecular biology labs. It is critical to quantify DNA input for assays to achieve quantitative and comparable results. Sometimes quantitation of DNA can be an important indicator or biomarker for disease progression and tumor burden, such as quantitation of circulating cell-free DNA (cfDNA).

Common DNA quntification methods often requires DNA extraction or purification to remove interfering components from matrix before DNA quantitation. These quantification methods include spectrometer methods, DNA-binding dye methods, and qPCR methods.

Drawbacks of Common DNA Quantification Methods

A big drawback for common DNA quantification methods is DNA extraction. This step is not only tedious for regular lab operators, it introduces experiemental errors both from the operator and extraction method itself. Therefore it is not uncommon  the DNA extraction step can lead 40 to 60% DNA loss varying between vendors and between extraction mechanims (such as beads vs. columns).

In addition, the downstream DNA quantification assays also bring errors from use of different methods, the use of different standards or references, even with different instruments for the same assay. Lack of standarization is the major problem about DNA quantification.

Introducing the Direct DNA Quantification Tool Powered by SuperbDNA™ Technology

SuperbDNA™ Technology is a modified branched DNA (bDNA) technology that more efficiently detects trace amounts of DNA in matrices without DNA extraction.

Rather than amplif target molecules which often needs DNA extraction, SuperbDNA™ technology captures the target and amplify chemical signals associated with the targets to directly and sensitively quantitate DNA without DNA extraction.

Platforms are developed for the Direct DNA Quantification Tool, based on SuperbDNA™ Technology

Applications are available for the Direct DNA Quantification Tool, based on SuperbDNA™ Technology

Introducing the 2 Platforms of Direct Human DNA Quantification Assays  

Luminometer-based platform

The luminometer-based platform: Captures probes are pre-coated at the bottom of 96 well-microtiter plates and chemiluminescent signals generated by alkaline phosphatase-substrate reaction are detected using luminometers.

Capture Probe Carrier: Pre-coated microtiter plates

Incubation Apparatus: Thermal incubator

Detection Instrument: Luminometer

VznHealth™ Luminometer-based Products

  • VznHealth™ Direct cfDNA Test
  • VznHealth™  Radiotherapy Toxicity Measurement Assay
  • VznHealth™ Direct Human Residual Host Cell DNA Assay

 

QuantiReader™ Benchtop Luminometer

Luminex-based Platform

The Luminex-based platform: Captures probes are conjugated to magnetic beads and signals generated from Streptavidin-PE are detected on Luminex MAGPIX.

Capture Probe Carrier: Magnetic beads

Incubation Apparatus: Thermal shaker

Detection Instrument: Luminex MAGPIX

QuantiDNA™ Luminex-based Products

  • QuantiDNA™ Direct cfDNA Test
  • QuantiDNA™  Radiotherapy Toxicity Measurement Assay
  • QuantiDNA™ Direct Human Residual Host Cell DNA Assay

 

Luminex MAGPIX

VznHealth™ and QuantiDNA™ DNA Quantification Assays Give Similar Testing Results

Comparison of DNA Quantitation Results: Luminometer-based Assay and Luminex-based Assay

Assay Linearity and Performance

The performance comparison of the two DNA quantitation platforms is summarized in the table on the right. Both platforms show the sensitivity that can measure low picogram of cfDNA in 20μl assay or very low ng/ml DNA. This range fits the healthy and cancer patient’s DNA quantitation with assay variation within 15%. In addition, no interference is found for the proteins or chemicals commonly found in plasma or reagents added to the plasma for DNA storage.

 

 VznHealth™ Direct cfDNA TestQuantiDNA™ Direct cfDNA test
Detection Limit 0.39 ng/ml (7.8 pg in 20 µl assay)0.09 ng/ml (1.8 pg in 20 µl assay)
Quantitation Range 0.39 to 50 ng/ml0.09 to 25 ng/ml
Intra-Assay Reproducibility<15%<10%
Inter-Assay Reproducibility<15%<15%
Interference from HemoglobinNoNo
Interference from cholesterolNoNo
Interference from EDTANoNo

The cfDNA Test Result Comparison

The cfDNA test result comparison for a cancer patient before and after radiation therapy using the QuantiDNA™ assay (bead-based) and VznHealth™ assay (plate-based) can be found in the figure on the right. Both VznHealthTM assay (plate-based) and QuantiDNATM assay (bead-based) test are used for measurement of cfDNA concentration change for one prostate cancer patient after 1 to 5 days of radiation treatment versus cfDNA levels before treatment (in absolute number or expression as fold change).

 

Direct Human DNA Quantification Applications

cfDNA Quantification

For research and clinical applications

Circulating Cell-free DNA (cfDNA) is a potential biomarker for different diseases such as cancer, cardiovascular diseases, and immune system and infection diseases. Directly monitoring the amount of cfDNA in plasma can be an effective way to monitor disease progression and prognosis.

Luminometer-Based Platform

VznHealth™ Direct cfDNA Test

Luminex-Based Platform

QuantiDNA™ Direct cfDNA Test

Radiation Therapy Toxicity Measurement

For research and lab-developed test (LDT) applications

Radiation therapy or radiotherapy is used for more than 50% of the cancer treatment and sometimes it is the only treatment. Most radiotherapy is a local treatment to kill tumor cells by breaking the tumor DNA. However, radiotherapy also damage the normal tissues, leading to adverse side effects resulting from the toxicity. Measuring the cfDNA level can assess the radiotherapy toxicity and provide useful clinical information.

Luminometer-Based Platform

VznHealth™ Radiotherapy Toxicity Measurement Assay

Luminex-Based Platform

QuantiDNA™ Radiotherapy Toxicity Measurement Assay

Residual Host Cell DNA Quantitation

For research and pharmaceutical impurity applications

Residual host cell DNA is regulated by different regulatory bodies in manufacture of biological products such as recombinant proteins, antibodies, and gene therapy lentivirus particles. Although multiple methods are used for host cell residual DNA quantification, DNA extraction is often required before the residual DNA from host cells is quantified. Residual DNA from human host cells such as HEK 293T or 293 cells can be detected and quantified using our direct DNA quantification assays.

Luminometer-Based Platform

VznHealth™ Direct Human Residual Host Cell DNA Assay

Luminex-Based Platform

QuantiDNA™ Direct Human Residual Host Cell DNA Assay

Resources

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