DNA Recognition Instruments
█ AGNIESZKA LICHANSKA
DNA recognition instruments allow rapid identification of the origin of DNA in an environmental or medical sample. Recognition of the source of DNA is important in pathogen (disease-causing agent) identification in public health surveillance, and diagnostic and military applications.
DNA recognition instruments utilize two main methods for DNA detection and identification, nucleic acid hybridization r polymerase chain reaction (PCR). Hybridization of nucleic acids allows differentiation of sequences that differ by as little as one base pair by using high temperature washes that remove partially matched DNA strands. Hybridization relies on the fact that single stranded DNA reforms a double stranded helix with a complementary strand. The method requires a single stranded target (unlabeled) and probe (labeled with a radioactive or fluorescent tag to detect signal). PCR-based detection in modern instruments is based on specificity provided by primers required for DNA amplification and fluorescent probes to detect the product in real time.
New technologies for DNA recognition. The standard methods used in diagnostics are not rapid enough for the immediate identification of pathogens in a case of a biological attack either on military personnel or civilians. Engineers and biologists, therefore, are designing new technologies to make DNA recognition rapid, robust, with increased sensitivity of the assays and improved identification of positive samples. Optical identification methods are primarily used in PCR-based instruments; however, new magnetic and electrochemical methods were developed for hybridization-based assays.
Hybridization-based technologies. Chip-based hybridization assays, where the target DNA is spotted onto a glass or plastic slide and a single stranded DNA probe is used to detect it, were developed recently by a number of companies. Technology allows placement of thousands of DNA molecules on the slide, but detection of the specific reaction is often lacking sensitivity. As a result, a number of research teams and commercial companies are researching better ways to identify a positive signal.
One breakthrough came with the implementation of electrical conductivity as a detection method. This method relies on the use of electrodes with gaps of 30–50nm in size, containing single stranded DNA molecules (oligonucleotides) immobilized on their surface (capture probes) and gold oligonucleotide nanoparticles allowing detection of electrical currents resulting from hybridization. Both oligonucleotides bind to the target sequence when the electrode is immersed in a solution containing target molecules. A modification of this method is the use of signal amplification by using a photographic solution as developed by a Northwestern University team. A salt wash before the addition of photographic developer removes mismatches and the silver coated gold particles can be easily visualized. The chip is then scanned using a flatbed scanner, removing the need for expensive equipment. This method is highly sensitive and very fast. It is able to detect concentrations of DNA (100 times more sensitive than conventional detection methods), in one to three minutes.
A modification of this method was developed in 2002 and incorporates nanoparticle probes that in addition to gold particles, have Raman dye-label (for example Cy3, Cy5, or Texas Red). Detection of these probes can be either by Raman spectroscopy or by using a flatbed scanner to detect silver enhancement. By using multiple labels one is able to design chips detecting multiple target sequences (multiple pathogens).
Hybridization-based instruments. The great advantage of hybridization-based instruments is the fact that they do not require any DNA amplification, are highly sensitive and give rapid results.
Scientists in industry are currently producing instruments that are based on measuring electrical conductivity. One is known as the eSensor. The system consists of bioelectronic chips, reader, and special software. The chips contain capture probes and signaling probes. After an interaction with a target sequence, signaling probes induce electric current, which is detected and interpreted by the sensor's software. This instrument can perform a number of assays simultaneously. A second instrument is directly based on the technology from the Northwestern University group, using a method of conductivity detection that was modified to amplify the signal from gold particles by using a photographic developer solution to coat the gold particles. Although this instrument currently requires a large space, work is underway to design a handheld device.
One company has licensed a Strand Displacement Amplification (SDA) method, and has devised an electrical method of binding DNA to silicon chips and performing hybridization. SDA oligonucleotides (probes) are localized to spots on the chip by charge and immobilized on the surface by chemical reaction. The sample is then added to the chip and by applying an electric current, the binding of test to the probes is highly accelerated (one to three minutes). By reversing the charge, unbound molecules are removed and only perfect matches remain. The entire process takes about 15 minutes. Chips for identifying pathogens such as the bacteria responsible for anthrax are under development.
PCR-based instruments. The newest technologies in polymerase chain reaction (PCR)-based instruments involve instrument miniaturization and methods for handling and detecting multiple pathogens in multiple samples. The ability to prepare clean PCR templates in a field is often difficult or limited. However, the presence of various chemicals can inhibit the amplification, giving false negative results and, in the case of an attempt to identify a biological threat, possibly endanger people's lives. As a result, a number of companies have started to offer sample preparation units with their PCR instruments.
The advanced nucleic acid analyzer (ANAA), developed in 1997, was the first DNA recognition instrument designed for work in the field. It was portable, but still large and was superseded by a hand-held ANAA (HANAA).
The major differences between the various instruments are in the proprietary heating and cooling systems, detection optics, and sample preparation and handling, as well as size. Speed of most of these instruments is similar with the typical sample analysis taking 7–20 minutes.
A different technology, but still PCR-based, uses a high-performance liquid chromatography to separate the PCR products and identify mutations. The advantage of the system is that it can detect mutations in any genes that could have been altered for designing biological weapons, thus, potentially complementing any other detection methods.
Application of DNA recognition instruments. DNA recognition instruments are likely to be used in general monitoring of the environment, investigation of suspicious objects, and in diagnostics. In all of these applications, detection must be rapid and accurate in order to introduce prevention measures or rapid treatment. Ease of use and result interpretation are important, as in majority of cases, users will be people with minimal laboratory training.
As of 2003, the majority of these advanced DNA recognition instruments were or are undergoing final testing in the field. They are able to cope with samples of water, food, and various clinical samples to detect an environmental contamination or identify a pathogen causing unusual symptoms in humans or domestic animals.
█ FURTHER READING:
PERIODICALS:
Belgrader, P., W. Bennet, D. Hadley, et al." PCR Detection of Bacteria in Seven Minutes." Science no. 5413: 449–450.
Cao, Y. W. C., R. Jin, C. A. Mirkin. " Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA detection." Science no. 5586 (2002): 1536–1540.
Park, S. J, T. A. Taton, and C. A. Mirkin. "Array-Based Electrical Detection of DNA with Nanoparticle Probes." Science no. 5559 (2002): 1503–1506.
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