Table of Contents > Genomics > Oligonucleotide microarrays in microbial diagnostics Print

Oligonucleotide microarrays in microbial diagnostics

Image

Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Bacteria, bacterial infection, chip, chromosome, diagnosis, disease, DNA, DNA microarray, fluorescence, hybridization, hybridize, microarray, microbe, oligonucleotide, plate reader, probe, RNA, mRNA, target.

Background
  • In humans and in all living organisms, DNA is packaged in structures called chromosomes. The DNA within the chromosomes contains genes, which provide the instructions for making the proteins that do the work in a cell. The instructions for making each protein are transferred to messenger RNA (mRNA). The mRNA carries the message to the cytoplasm, the part of the cell where the proteins are made. Each gene produces its own unique mRNA. The amount of different mRNAs in a cell is an indicator of how much of each protein encoded by a particular gene is produced by that cell. All of the active genes in a cell, determined by identifying all of the mRNA present within a cell, is called a transcriptome.
  • DNA microarray technology allows researchers to measure the amount of mRNA in a biological sample, such as cells or a tissue. DNA microarrays allow researchers to simultaneously measure the amount of thousands of mRNAs at the same time, which may greatly speed up experiments.
  • DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. Each short piece of DNA attached to a microarray is called an oligonucleotide probe (or a probe, for short). When using microarrays to study bacteria, researchers design probes that are able to recognize bacterial genes. Every probe is different and attaches specifically to one gene, and microarrays are often designed so that they contain enough probes to detect hundreds or thousands of different genes. If a microarray contains a probe for a specific gene, then it can be used to check for the presence of that gene in a sample.
  • Researchers may use microarray technology to study whether bacteria are present in a specific sample. For example, microarray technology may be used to help diagnose a sick individual with a bacterial infection. A blood sample may be taken from that individual, and testing the mRNA from the blood sample on a microarray could demonstrate a bacterial infection (if bacterial mRNA is detected by the microarray). If researchers design a microarray so that it has probes for multiple bacterial species, researchers can test for the presence of many different species of bacteria at once.

Methods
  • Design microarray: DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. Each short piece of DNA that is attached to a microarray is called an oligonucleotide probe (or a probe, for short). Every probe is different, and microarrays are often designed so that they contain probes that are able to detect hundreds or thousands of different mRNAs. When using microarrays to study bacteria, researchers design probes that are able to recognize bacterial mRNAs. By designing probes that can detect mRNAs from different strains of bacteria, researchers can use the same microarray to check for the presence of multiple bacteria strains in the same experiment. Probes on a microarray are arranged in rows and columns across the slide in known locations, so that researchers can determine which probe is used to detect a specific mRNA. Probes are usually attached to the microarray slide with the help of robotic machines.
  • Microarrays, such as GeneChip® (manufactured by Affymetrix), which contain probes to detect thousands of human mRNAs, are manufactured and sold commercially. Researchers may also produce their own microarrays. This may be less expensive, especially if a researcher is interested in studying a smaller number of mRNAs and needs a microarray with only enough probes to detect these mRNAs.
  • Prepare sample: Microarrays are commonly used to detect the level of mRNA in cells. To perform a microarray experiment, mRNA from cells or tissues of interest is obtained; the sample mRNA is called the target. Because cells contain more than just the sample mRNA, the mRNA needs to be purified and separated from the other components in the cell. The mRNA is then attached to a fluorescent dye. In some cases, researchers may study two different samples at once. In this type of experiment, two different colored dyes are used, and mRNA from each sample is attached to a different colored dye.
  • Hybridize the sample: The labeled target is then put onto the microarray. If a probe designed to detect a specific piece of target mRNA is present on the microarray, then that mRNA will stick to the microarray. The more mRNA that is present in the target sample, the more of it will stick to the microarray. The process of letting the target stick to the probe is called hybridization. After the target is given a chance to stick to the microarray, the target that did not stick is washed off.
  • Detect sample: Researchers may measure how much of a specific mRNA was present in the biological sample by measuring the amount of fluorescent signal produced by the target that remained stuck to the microarray. Each probe on a microarray is in a known location, and if the probe for a certain gene has a strong fluorescent signal, then a large amount of mRNA from that gene was present in the biological sample. If a certain probe produces a low (or no) fluorescent signal, then mRNA corresponding to that gene was not present (or was present in low amounts) in the biological sample. The fluorescent signal is determined using a special machine called a fluorescent plate reader that can detect and measure the strength of fluorescent light.
  • Because the probes on a microarray are organized in a pattern of rows and columns, the results from a microarray experiment typically look like a pattern of colored rows of dots. Dots with a very bright color correspond to probes that were able to detect the target, whereas dots without any color correspond to probes that did not detect the target in the sample. In some microarray experiments, two different target samples are used, each labeled with a different colored dye (such as red or green). In these types of experiments, each sample may contain specific mRNAs that hybridize to the same probe. The difference in the amount of mRNA between the two samples can be determined by measuring the ratio of the two different dyes in a specific spot on the microarray. When two different colored dyes are used, they may make a new color, and the intensity of this new color can be measured.

Research
  • Diagnose diseases: Microarray technology may be used to help diagnose a sick individual with a bacterial infection. A fluid sample, such as blood or saliva, may be taken from that individual, and testing the mRNA from the sample on a microarray could show a bacterial infection if bacterial mRNA is detected by the microarray. If researchers design a microarray so that it has oligonucleotide probes (short sequences of DNA designed to recognize a target) for multiple bacterial species, researchers can test for the presence of many different species of bacteria at once.
  • For example, bacteria that commonly cause respiratory tract infections include Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae, and Mycoplasma pneumonia. Researchers have designed a microarray with oligonucleotide probes that detect DNA or mRNA from these bacterial species. By testing a biological sample, such as a throat swab or saliva, from an individual with a respiratory tract infection, the results from this microarray could alert doctors to the presence of one of these bacterial species in a patient. Identification of the cause of a respiratory tract infection would likely lead to more successful treatment of the infection and would decrease the use of ineffective therapies.
  • Identifying antibiotic-resistant bacteria: Bacterial infections are commonly treated with antibiotics, which are drugs that kill or inhibit the growth of bacteria. However, sometimes bacteria undergo genetic mutations and become resistant to certain antibiotics. In these cases, the use of antibiotics to fight the bacterial infection may be unsuccessful. Therefore, it is valuable to determine whether an individual has been infected with bacteria that are resistant to antibiotics.
  • Microarrays may be used to detect antibiotic-resistant bacteria. In these cases, oligonucleotide probes are designed to detect the genes that are known to lead to bacterial resistance. By applying a sample of bacterial DNA or mRNA to the microarray, researchers may determine whether the bacteria contain any antibiotic-resistant genetic variants. For example, the bacteria Bacillus anthracis, which causes anthrax, may be treated with antibiotics such as ciprofloxacin (Cipro®), doxycycline (Vibramycin®), rifampin (Rifadin®), and vancomycin. Due to mutations in specific genes, however, Bacillus anthracis may become resistant to these antibiotics. Researchers have developed a microarray that contains probes for the genes involved in antibiotic resistance, which may be used to determine whether individuals are infected with antibiotic-resistant bacteria.

Implications
  • More rapid diagnoses: Microarrays may be designed to detect the presence of dozens of bacterial strains in the same experiment. Microarrays can detect bacterial target DNA with oligonucleotide probes, short sequences of DNA designed to recognize a target. This may allow clinicians to more quickly determine which specific strain of bacteria is infecting a patient. Other methods that were used before microarrays to genetically test for bacteria, such as polymerase chain reaction (PCR), typically cannot test for a large number of strains in the same experiment. PCR is a method that uses proteins called enzymes to amplify and detect genes.
  • More effective treatments: Microarrays may alert clinicians in cases in which a patient is infected with antibiotic-resistant bacteria. If antibiotic-resistant bacteria are detected, clinicians may make a more informed decision about treatment options and choose not to administer antibiotics that would likely be ineffective.
  • Improved quality of life: By leading to more effective treatment through improved diagnosis, microarrays may improve the quality of patients' lives. Patients may have a shorter duration of diseases, may need to spend less time at their doctors' offices, and may miss fewer days at school or work.

Limitations
  • Microarrays may be used to detect the expression of hundreds or thousands of genes in just one experiment. In order to detect a specific mRNA, however, researchers need to have prior knowledge of the mRNA and the mRNA sequence to design an oligonucleotide probe, a short sequence of DNA designed to recognize a target, for that mRNA. Therefore, researchers cannot use microarrays to discover new mRNAs or to diagnose diseases in which the causative gene is unknown. Instead, microarrays are used to detect mRNAs that have already been characterized.
  • Researchers must also be careful when designing oligonucleotide probes to use for microarray analysis. An oligonucleotide probe is a sequence of DNA that can recognize and bind to the mRNA, or to a region of the mRNA, that a researcher is interested in studying. Some regions of one mRNA may be very similar to regions on a different mRNA. Therefore, researchers may need to generate an oligonucleotide probe or primer specific to the mRNA they are interested in, but that is not similar to other mRNAs. Otherwise, researchers may think they have identified the mRNA of interest when they have really identified a different but related mRNA. This is called a false-positive result.
  • Microarray experiments may be expensive to perform, so cost may be a drawback to using them.

Safety




Future research
  • In the future, researchers may develop microarrays that contain oligonucleotide probes, short sequences of DNA designed to recognize a target, for larger numbers of different bacterial strains, allowing scientists to detect more strains of bacteria. As researchers continue to discover new genes in different bacterial strains, they learn more about the genetic information of these bacteria, which will allow them to design oligonucleotide probes to detect these strains.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
  1. Antwerpen MH, Schellhase M, Ehrentreich-Förster E, et al. DNA microarray for detection of antibiotic resistance determinants in Bacillus anthracis and closely related Bacillus cereus. Mol Cell Probes. 2007 Apr;21(2):152-60.
  2. Jayapal M, Melendez AJ. DNA microarray technology for target identification and validation. Clin Exp Pharmacol Physiol. 2006 May-Jun;33(5-6):496-503.
  3. Lodes MJ, Suciu D, Wilmoth JL, et al. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray. PLoS ONE. 2007 Sep 26;2(9):e924.
  4. National Center for Biotechnology Information. .
  5. National Human Genome Research Institute (NHGRI). .
  6. Natural Standard: The Authority on Integrative Medicine. .
  7. University of Arizona Department of Molecular and Cellular Biology. .
  8. Wiesinger-Mayr H, Vierlinger K, Pichler R, et al. Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition. BMC Microbiol. 2007 Aug 14;7:78.
  9. Zhu H, Cabrera RM, Wlodarczyk BJ, et al. Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity. BMC Dev Biol. 2007 Nov 20;7:128.

Copyright © 2011 Natural Standard (www.naturalstandard.com)


The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

Search Site