Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-03-03
2002-10-01
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06458584
ABSTRACT:
A novel microchip is customized to answer specific questions and has oligonucleotides positioned on the microchip so that multiple bits of information are evidenced to a simpler pattern A new method of hybridization to a microchip is also presented.
BACKGROUND OF THE INVENTION
Differences in nucleotide and amino acid sequences may be exploited to analyze environmental, food or biological samples. Detection and identification of microorganisms is important for clinical purposes and for determination of contaminated food, air, water or soil. Studies in environmental microbiology are often limited by the inability to unambiguously identify and directly quantify the enormous diversity of natural populations. This problem is now changing with increasing use of molecular techniques to directly measure different genetic features. (Mobarry et al., 1996; Stahl, 1995; Wagner et al., 1995) For example, DNA probes are now commonly used to detect by hybridization, genes encoding proteins involved in specific catabolic functions, and to resolve different genetic populations in the environment. In particular, the use of group-specific DNA probes complementary to the small subunit (SSU) 16S rRNA has provided a comprehensive framework for studies of microbial population structure in complex systems. Sequencing of this subunit revolutionized microbial classification and led to the discovery of archebacteria. (Woese, 1987) A large number of the sequences for different organisms has been collected. (Maidak et al., 1996) Every microorganism species is characterized by a specific DNA sequence within a variable region of its ribosomal RNA gene or other genes. A highly efficient procedure for microorganism classification and for construction of their evolutionary trees is based on these observations. Identification of specific sequences in ribosomal DNA is a reliable microbial analysis that can be carried out by direct DNA sequencing. However DNA sequencing is a rather complicated, expensive and time consuming procedure to use for serial microbial analysis on a commercial scale for environmental or medical applications. Consequently, new methods are needed to make sequence matching commercially feasible.
Also, methods are needed that are transportable to the field. A nucleic acid hybridization is a highly specific and sensitive procedure that allows a specific sequence to be detected and identified among other millions of sequences in a genome of higher organisms, or among a mixture of different organisms. The principle of hybridization is that sequences hybridize as a function of the similarity of their linear nucleotide sequence. The hybridization of DNA or RNA extracted from even a very complicated mixture to a specific oligonucleotide probe has resulted in unambiguous identification of specific microorganisms in an environmental sample, for example. In the course of such an analysis, RNA or DNA is extracted from a sample of microorganisms isolated from water solutions, air or soil, immobilized on a filter and then hybridized successively with several oligonucleotide probes for different microorganisms. However, for this purpose, the sample needs to be checked for the presence of hundreds or thousands of different oligonucleotides corresponding to various microorganisms which is prohibitively laborious and expensive using present methods and yields results that must be interpreted by a computer in order to decipher the identification. What is needed is a simplified pattern to provide rapid answers to specific questions, e.g. are any known pathogens in a water sample?
The scope of applications of nucleotide hybridization is often limited by the nature of the assays, generally involving the independent hybridization and interpretation of multiple environmental samples to multiple DNA probes. In addition, some detection assays require amplification of the target nucleic acid, for example, via PCR. This may contribute to quantitative biases. Thus, there is need for assays that provide for greater sample through-put capacity and greater sensitivity, rapid read-out of results.
Another area in which specific DNA or RNA sequences are of interest is mutation and polymorphism analyses. The number of base changes discovered (mutations) in different genes is growing rapidly. These changes are associated with genetic diseases, with disease predispositions and cancers, with development of drug resistance in microorganisms, and with genetic polymorphisms. Polymorphisms are useful for determining the source of a sample, e.g. in forensic analyses. Polymorphisms such as in the HLA system are essential to predict success of tissue transplants. The ability to simultaneously analyze many mutations in a gene in a simple, fast, and inexpensive way is essential in clinical medicine and this need has stimulated the development of different methods for screening mutations, but all have serious limitations. What is needed are kits that are transportable and interpretable, e.g. for use in clinics without high technology microscopes.
Hybridization of filter-immobilized DNA with allele-specific oligonucleotides was suggested as a way to screen for mutations. (Conner et al., 1983) However, the number of alleles that can be assayed at one time is limited, the filters are usable only for a few times, and there is little opportunity for complex analysis or easy interpretation of results.
A possible solution to large scale hybridization is to use microchips for DNA sequence hybridizations (SHOM, sequencing by hybridization with oligonucleotides in a microchip) (e.g. Khrapko, 1996; Yershov, 1996). The development of an array of hundreds or thousands of immobilized oligonucleotides, the so-called “oligonucleotide chips”, permits simultaneous analysis of many mutations (for a review, see Mirzabekov, 1994). Such arrays can be manufactured by a parallel synthesis of oligonucleotides (Southern et al., 1992; Fodor et al., 1991; Pease et al., 1994; Matson et al., 1995) or by chemical immobilization of presynthesized oligonucleotides (Khrapko et al., 1991; Lamture et al., 1994; Ghu et al., 1994). Glass surfaces (Southern et al., 1992; Fodor et al., 1991; Ghu et al., 1994), glass pores (Beattie et al., 1995), polypropylene sheets (Matson et al., 1995), and gel pads (Khrapko et al., 1991; Yershov et al., 1996) have been used as solid supports for oligonucleotide immobilization. However “Oligonucleotide array technology has not yet lived up to its promise.” Southern, 1996 p. 115.
Some of the deficiencies in the art are unpredictability of the results, lack of knowledge of optimum conditions, and failure to demonstrate accuracy and commercial feasibility. Moreover, analysis of the results of hybridization requires computer programs capable of assimilating and interpreting multiples bits of information, and high technology microscopes. The microchips are neither portable, reusable, nor easily interpreted.
SUMMARY OF THE INVENTION
This invention embodies applications of oligonucleotide microchip technology wherein the microchip is a biosensor and customized oligonucleotide microchips are designed for specific applications of nucleic acid hybridization.
Hybridization is a process by which, under defined reaction conditions, partially or completely complementary nucleic acids are allowed to join in an antiparallel fashion to form specific and stable hydrogen bonds.
Aspects of the invention include:
1. microchips designed so that multiple bits of genetic information are converted to a pattern, which is interpreted as a unit, wherein the appearance of the pattern provides answers to specific questions; this construction facilitates providing easily interpretable answers provided by hybridization patterns and removes some need for high technology instruments to interpret the results of hybridization; and
2. improved methods of hybridizing oligonucleotides in a sample to oligonucleotides on a customized microchip do not require a washing step but rather measure non-equilibrium melting curves (temperature curves) that do not require washing with
Chik Valentine
Drobyshev Aleksei
Fotin Alexander
Guschin Dmitry Y.
Lysov Yuri
Barnes & Thornburg
Martin Alice O.
Siew Jeffrey
University of Chicago
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