Simple and portable method for DNA detection
This technology is a simple, sensitive and highly portable enzyme-based optical method to detect DNA produced by polymerase chain reaction (PCR) or other DNA amplification methods. It is a flexible platform that can be used for end-point or real-time monitoring of PCR products using benchtop thermocyclers or microfluidic devices in both homogeneous and heterogeneous formats.
The global market for genetic testing is expected to reach $2.2 billion by 2017, and it represents the most rapidly expanding segment of the molecular diagnostics market. Increasing knowledge about potential benefits of genetic testing, diagnosis of rare and fatal disorders, predicting the probabilities of occurrence of diseases, and tailoring personalized medicine for specific patient populations all contribute to market growth. Wide adoption of genetic testing creates a great demand for low-cost, miniaturized, reliable DNA detection devices and reagents.
Enzyme amplified redox-based colorimetric DNA detection system
Researchers at the University of Michigan developed a DNA detection platform that uses a labeled sequence-specific DNA probe, a PCR reagent mixture, and an enzyme-based optical detection system. The cofactor labeled site-specific probe hybridizes to its complementary DNA sequence, and during PCR exonuclease activity of DNA polymerase cleaves away this cofactor. Once released from the DNA probe, the cofactor binds to and activates the enzyme in the detection system. Cofactor-activated enzyme catalyzes a reaction that leads to reduction of a mediator molecule, and a redox-sensitive dye then reoxidizes this mediator. As a result, color change of the dye is directly correlated to the amount of target DNA sequence present in the sample.
This DNA detection methodology was tested with a site-specific DNA probe against P. pachyrhizi plant pathogen conjugated to an enzyme cofactor. Glucose dehydrogenase enzyme was used as a cofactor-dependent signal generating enzyme, in combination with three different redox-sensitive dyes. Changes in absorbance were proportional to the concentration of soluble cofactor or PCR product in the sample, and sub-nanomolar concentrations of cofactor produced a robust signal.
The enzymatic signal amplification step greatly enhances assay sensitivity and enables miniaturization of this platform, and a colorimetric readout allows use of an inexpensive diode light source in the detector. Non-specific signal originating from enzyme activation by an intact cofactor-DNA probe can be eliminated by chemically introducing a steric hindrance, addition of a size-exclusion matrix that will only allow small molecules to reach the enzyme, or by adding poly-cations that will sequester the uncleaved probe. These simple modifications offer excellent signal-to-noise profiles and can be adjusted according to specific application.
Genetic tests are used in various industries including healthcare, agriculture, food testing, and bio-defense. They can be used to diagnose a wide range of conditions, including cancer, genetic markers associated with a high risk of cancer, Alzheimer’s disease, genetic diseases (e.g. cystic fibrosis), as well as detect a variety of infectious diseases including anthrax, HIV, and tuberculosis. DNA diagnostic devices range from standard benchtop apparatuses to microfluidic lab-on-a-chip setups and DNA test strips. A fully integrated system that is capable of automating cell lysis, nucleic acid extraction, PCR set-up, amplification and detection with minimum human intervention will greatly reduce cost, time and labor for these tests. The primary bottleneck for such a system is that detection instrumentation remains rather bulky and expensive. Majority of PCR detection reagents utilize fluorescence due to its high sensitivity. Products such as TaqMan, Molecular Beacons, and Scorpions use two fluorophores, and are expensive to manufacture. SYBR Green is a cheaper alternative, but it cannot discriminate between specific and non-specific DNA sequences and still requires expensive fluorescence detection optics. Enzyme amplified DNA detection technology is a low-cost, sensitive, robust, simple, and portable solution for multiple genetic testing applications.
Applications and Advantages
- DNA detection platform for genetic testing applications
- Can be used in healthcare, food testing, agriculture, and bio-defense
- Low-cost reagents and instrumentation
- Utilize low-cost diode light sources
- Highly portable
- Sensitive and specific DNA detection
- Simple to use