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September 26, 2000
Olympus Introduces
Oligonucleotide Probe Sequence Design Software
for Genetic Analysis
- Revolutionary technology for pioneer research in the post-genome era -
Olympus Optical Co., Ltd. (President: Masatoshi Kishimoto) and Assistant Professor Akira Suyama of the University of Tokyo, Graduate School of Arts and Sciences have jointly developed sequence design software for oligonucleotide probes*1 capable of high-speed calculation of sequences. This new technology is expected to revolutionalize the genetic analysis of cancer and genetic diseases among others in the new era of genetic medical treatment. With the conventional method*2, highly specific sequence meant that the calculation took enormous amount of time from weeks to even months. The unique algorithms used to develop this software dramatically accelerate the process, allowing finer sequences to be calculated more accurately in just a few days. The new software may provide an immediate solution to the global quest for a high-speed detection and analysis of target genes among huge sequences.
To date, the human genome project has resulted in the identification of as many as approximately 3 billion bases of complete human genomic sequences. As we enter the post-genome era, the focus is now shifting to the task of applying base sequencing technology to medical purposes. This work is expected to result in the identification of genes, whose number is estimated to be approximately 100,000, that are linked to lifestyle diseases such as diabetes, or to cancer. It will then be possible to develop advanced genetic diagnosis technology, including disease risk diagnosis methods for use in disease prevention, and drug response diagnosis methods*3 that will contribute to the effective administration of medication.

Genetic analysis involves the reading of base sequences in DNA*1 extracted from tissue or blood. This is accomplished by a technique known as hybridization*1 using DNA microarrays*4. Single-stranded DNA oligonucleotide probes ("oligo probes") consisting of 20-30 bases are employed for this purpose. The oligo probe is designed to complement the sequence of the target gene. However, it is difficult to select a sequence that will specifically hybridize only to the target, and success depended on the intuition and experience of researchers. Unfortunately, some oligo probes would end up combining with other genes, and it was necessary to identify the appropriate sequence through experimentation.

Our new oligo probe design software uses the tuple method*5 developed by Assistant Professor Suyama. This method is capable of designing a highly specific oligo probe sequence without the need to scan the target genome many times. Moreover, the software consists of filters*6 for probe selection. This ensures the creation of an optimal sequence that is grouped at an appropriate melting temperature and does not form intramolecular structures*7. By using these oligo probes, which have uniform melting temperature, it will be possible to conduct tests under different temperature conditions and achieve enhanced precision in genetic diagnosis with the DNA capillary array system*8 now being developed by Olympus.
* 1 DNA Characteristics and Hybridization Method- Oligo Probes
(1) DNA (deoxyribonucleic acid) is a molecule that carries the genetic information of living organisms. It consists of a sugar-phosphate backbone and a nitrogenous base. DNA can be divided into four types according to the nitrogenous base type. Among these, adenine (A) and thymine (T) as well as guanine (G) and cytosine (C) are complementary. The double stranded structure is formed through the hybridization of these complementary single stranded chains.
(2) Given these characteristics, it is possible to detect a target gene by hybridizing it with DNA that has a complementary sequence. This method is known as the "hybridization method." An oligo probe is a name of the DNA with a length (oligo) of several to several dozen bases. It is used as a detection probe.
(3) The hybridization between the two strands breaks down, leaving two separate single strands, when the temperature of the solution containing the DNA rises. This melting temperature depends on the sequence type. To achieve precise hybridization, it is necessary to conduct experiments under conditions that correspond to the melting temperature of the oligo probe.
* 2 Conventional Method for Checking Target Specificity
All candidate sequences were cross-checked to ensure that hybridization occurred only at the target locations.
Checks are carried out at each base location in the sequence to ensure that hybridization does not occur with any gene other than the target. The sequence shown on the left is unsuitable as a candidate, since the possibility of mis-hybridization is extremely high.
It is very time-consuming to apply this cross-checking to all candidates. To carry out the calculations needed to identify candidate sequences for organisms with long genetic sequences such as humans or mice, it is necessary to use a super or special-purpose computer.
* 3 Drug Response Diagnosis
Just as there are people who are resistant or susceptible to the effects of alcohol, so the efficacy of drugs varies according to the individual. In the case of anticancer drugs and others with strong side effects, knowing in advance whether or not a particular drug will work can help to avoid unnecessary suffering for the patient.
*4 DNA Microarrays
A DNA microarray is a sensor device that detects numerous genes through the hybridization process. They are created by fixing oligo probes for example, to a spot with a diameter of several hundred microns at specific locations on a glass slide or other substrate. A sample solution containing target nucleic acids, which are labeled with fluorescent substances, is then applied to these spots. If hybridization occurs with the target, the locations in which fluorescence occurs can be used to determine the presence of specific kinds of genes in the sample.
* 5 The Tuple Method
(1) A tuple is a short sequence consisting of several bases. (The example shown here has seven.) The tuple method involves the counting of the frequency that a given tuple appears among all genes. This counting process is carried out only once at the beginning.
(2) The tuples contained in the candidate sequence are then checked to determine if there are any that appear frequently. The specificity is then calculated to assess whether or not the candidate is a "frequent" sequence in the entire gene.
* 6 Composition of Software and Calculation Filters
Once the tuple frequency has been calculated, candidate sequences with high specificity are selected through tuple assessment. After applying certain restrictions derived from the experimental conditions, these are then passed through filters to narrow the range of candidates with thermal properties suitable for the proposed experiment.
* 7 Intramolecular Structures
DNA tends to undergo complementary hybridization. Given a sufficient strength, even a single-strand DNA molecule may form an intramolecular structure, thus suppressing hybridization. The potential for the formation of such intramolecular structures can be predicted through calculations.
* 8 Using Oligo Probe DNA Capillary Arrays Designed with this Software
(1) A DNA capillary array is a gene detection device integrating oligo probes fixed at specific locations in capillaries that are complementary to the target gene.
(2) The reaction conditions can vary according to each capillary, so it is possible to achieve hybridization reactions simultaneously under conditions suitable for each target, simply by putting oligo probes designed with this software into different capillaries with each temperature setting.
*Olympus Optical Co., Ltd. was changed to OLYMPUS CORPORATION as of October 1, 2003.
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