Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-30
2001-05-08
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06228580
ABSTRACT:
This application is a 371 of PCT/FR96/01201 filed Jul. 30, 1996.
The present invention relates to a method for detecting nucleic acids using nucleotide probes permitting both specific capture and detection. The present invention permits automation of the large-scale specific detection of nucleic acids using a single-stranded ribonucleotide probe complementary to a target nucleotide sequence, said probe permitting both capture on a suitable surface and rapid and sensitive detection.
As a result of the major genome sequencing and analysis programmes, biology some years ago entered a new era, that of the massive acquisition of data based on the intensive use of improved existing techniques. In five to ten years time, the genome of several model organisms, as well as the human genome, will be completely sequenced. Since the number of genes encoded by the human genome is calculated at between 50,000 and 100,000, several thousand sequences of coding regions (cDNA) and also of regulatory regions (promoters) will have to be listed, analyzed and integrated in databases, and their expression profiles in some hundred tissues or cells will be able to be established.
The expression profile of a gene consists in studying the relative abundance of the corresponding messenger RNA (mRNA) expressed in particular cells (for example belonging to different tissues, or representing different stages of development, particular pathologies, induction by drugs, and the like). Establishment of the expression profiles of genes hence requires the specific and sensitive detection of mRNAs.
The detection of specific mRNAs may be undertaken by two different types of approach: those based on amplification techniques and those based on hybridization techniques.
The use of amplification techniques such as RT-PCR (reverse transcriptase-polymerase chain reaction) appears to be of limited applicability in a planned large-scale study of mRNA expression profiles on account of the cost which would be entailed in synthesizing the large number of oligonucleotides needed for carrying it out (for 50,000 different genes, the minimum number would be 100,000 oligonucleotides).
The “sandwich” hybridization technique described for the first time by Dunn A. R. and Hassell J. A. (1977, cell, 12, 23-36) was developed in order to avoid the purification and immobilization of target nucleic acids which formerly necessitated detection after solid-phase hybridization. This technique is based on the existence of two nonoverlapping probes directed against the same target nucleic acid. The first probe is immobilized on a solid surface and permits capture of the target. The second probe possesses a tracer in its sequence and permits detection of the target. This technique is essentially used for the detection of amplification products. In actual fact, the probes used are small (from 20 to 25 nucleotides) and, unlike long probes, they do not enable strong hybridization signals to be obtained. This technique is unsuitable for the simultaneous detection of a large number of targets, since each probe necessitates hybridization temperatures which can be different.
The second approach is based on hybridization techniques that do not involve amplification, and which hence require great sensitivity. There are several formats which may be used to detect a specific hybridization: liquid-phase hybridization; solid-phase hybridization; in situ hybridization on tissues or cell bodies. In all cases, the probe is a nucleic acid (DNA or RNA) capable of hybridizing with a complementary nucleic acid sequence: the target (DNA or RNA). The kinetics of the different nucleic acid hybridization reactions are well known (Britten et al., 1974, Methods Enzymol, 29, p. 363; Kohme et al., 1977 Biochemistry, 16 pp. 5329-5341), and knowledge of the parameters involved in the rate of hybridization as well as in the stability of the target-probe hybrids enables the optimal conditions to be determined for obtaining the best signal-to-background ratio.
Liquid-phase hybridization is very efficient and has the advantage of affording the fastest rate of hybridization. However, it is difficult to separate the free probe from the hybridized probe. Solid-phase hybridization methods which involved immobilization of the target (or of the probe) on a solid surface (e.g. nitrocellulose, nylon) are easier to carry out from the standpoint of separation of the unhybridized probe. However, compared to hybridization in solution, the rate of hybridization is from 7 to 10 times as slow. Lastly, in situ hybridization permits the microscopic examination of DNA or RNA sequences present in cells or tissues while preserving their localizations. This method is suited to cytology or histology laboratories.
In the specific case of the detection of RNAs, the so-called “northern blot” solid-phase technique constitutes the most widely used hybridization method, used both in medical analysis and in basic research. Northern blotting, while it is the only method capable of providing information about the size of messengers, and hence about the identity of the target, cannot be considered for large-scale analyses because of its low sensitivity and difficulties of automation. For similar reasons, the in situ hybridization technique likewise appears to be unsuitable for large-scale studies of RNA expression profile.
Among the methods of detection of RNA based on hybridization in solution, the nuclease protection technique has the advantage of being a very sensitive and specific method. In this technique, single-stranded RNA or DNA radioactive probes are hybridized in solution with RNA preparations containing the target RNA. After hybridization, the unhybridized nucleic acids (both the unhybridized probe and the RNAs of the preparation) are degraded by the action of nucleases specific for single-stranded nucleic acids (in general, RNases A and T1 for RNA probes, and nuclease S1 for RNA or DNA probes). The hybridization of the probe with its complementary RNA target “protects” the probe from degradation by the nuclease, and results in double-stranded molecules whose length is defined by the probe-target complementarity.
On the other hand, nonspecific hybridization of the probe with RNAs other than the target results in double-stranded molecules which are shorter than those originating from the specific hybridization. The two types of products, specific and nonspecific, may hence be distinguished according to size, the most usual analysis being polyacrylamide gel electrophoresis. An alternative technique of analysis by chromatographic separation has been proposed in WO 95/113116.
While the nuclease protection technique has the advantage of being sensitive and specific, the analytical method consisting of an electrophoretic or chromatographic separation is incompatible with automation of the method and use of a large number of samples.
The present invention is designed to permit specific detection of nucleic acids, in particular of RNA, in a format which makes it possible to handle simultaneously a large number of samples by a procedure which can be fully automated, while being sensitive, specific a reproducible.
Another object of the present invention is, in particular, to provide a method which permits the specific detection of mRNAs from total RNAs.
The invention is based on the use of a method of hybridization in solution of nucleic acids with a nucleotide probe which, in addition to a specificity of recognition brought about by its sequence, possesses modifications which enable it to accomplish two different functions. The first function permits binding to a solid support of the probe-target hybrid, or of the probe originating from a previously denatured probe-target hybrid. The second function of the probe permits specific detection by radioactive or cold detection methods.
Generally speaking, the invention involves a methodology comprising:
(1) a hybridization in the liquid phase using a doubling labeled nucleic acid probe that hybridizes specifically with a given RNA or DNA.
(2) a degradation by nuc
Blumenfeld Marta
Bouillot Michel
GENSET
Knobbe Martens Olson & Bear LLP
Whisenant Ethan
LandOfFree
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