DNA Recognition Instruments
DNA recognition instruments (Forensic Science)
The detection of DNA (deoxyribonucleic acid) at a crime scene begins with the identification and isolation of biological evidence such as blood, semen, saliva, or hair. Historically, such evidence has been found through physical searches of crime scenes, but the search process has been expedited by the development of specialized light sources and chemical tests.
Most commonly, the detection of biological samples has been aided by the use of ultraviolet (UV) light sources. UV lights belong to a class of instruments known as alternate light source (ALS) instruments. Unlike light sources that emit wavelengths of light across a broad spectrum, ALS lights use filters or special bulbs to emit a much narrower range of wavelengths. For example, UV lights emit wavelengths in the 400-200 nanometer range. The most common UV light is called a “black light,” which emits wavelengths in the 400-320 nanometer range, also known as UVA. These wavelengths are invisible to the human eye but may cause certain chemicals, mainly proteins, to fluoresce. UV lights are useful in the detection of blood, semen, and saliva. Other ALS instruments include copper and argon lasers and modified arc lamps. The use of lasers in DNA detection is becoming increasingly popular because lasers do not damage DNA strands and can emit very specific wavelengths of light.
ALS instruments can indicate fluorescing molecules, but upon finding such samples, investigators must...
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Further Reading (Forensic Science)
Butler, John M. Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers. 2d ed. Burlington, Mass.: Elsevier Academic Press, 2005
James, Stuart H., and Jon J. Nordby, eds. Forensic Science: An Introduction to Scientific and Investigative Techniques. 2d ed. Boca Raton, Fla.: CRC Press, 2005.
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DNA Recognition Instruments (World of Forensic Science)
With the advent of molecular detection techniques, the repertoire of forensic tools has grown considerably. The ability to detect deoxyribonucleic acid (DNA) and even to match the nucleic acid to its source can allow the forensic scientist to identify an individual, or to determine if the individual was at the scene of a particular investigation.
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 various public health, diagnostic, and military forensic applications.
DNA recognition instruments utilize two main methods for detection and identification. These are nucleic acid hybridization and the 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 the specificity provided by primers required for DNA amplification and fluorescent probes to detect the product in real time.
Engineers and biologists are designing new technologies to make DNA recognition rapid and 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.
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 300nm 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.
Scientists at Northwestern University produced a modification of this method, called signal amplification, using a photographic developing solution. 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 further modification of this method was developed in 2002. It 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).
The great advantages of hybridization-based instruments are 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 hand-held 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 the 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.
The newest technologies in 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. 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. The speed of most of these instruments is similar to the typical sample analysis taking 70 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.
DNA recognition instruments are likely to be used in general monitoring of the environment, investigation
SEE ALSO Analytical instrumentation; Biological weapons, genetic identification; Chemical and biological detection technologies; DNA profiling; RFLP (restriction fragment length polymorphism); STR (short tandem repeat) analysis.