Process for detecting nucleic acids by mass determination

Details Process for detecting nucleic acids by mass determination Process for detecting nucleic acids by mass determination

1999-03-17

2001-05-22

Horlick, Kenneth R. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S094000

Reexamination Certificate

active

06235476

ABSTRACT:

The invention concerns a method for detecting nucleic acids based on their mass and a reagent suitable for carrying out this method.
Nucleic acids are being used more and more in analytics as an indicator for the presence of microorganisms or for the genetic state of an organism. The reason for this is the amount of information which is contained in nucleic acids. The detection of mutations at the nucleic acid level is considered to be useful especially in the field of diagnosing genetic diseases.
In one type of nucleic acid detection the hybridization of labelled probes with the nucleic acids to be detected is evaluated as an indication for the presence of the nucleic acid to be detected. These tests can be carried out heterogeneously as well as homogeneously. In heterogeneous assays, after a sample preparation step the isolated target nucleic acid is usually amplified by a nucleic acid amplification method such as e.g. PCR according to EP-B-0 200 362, it is then immobilized on a solid phase and subsequently detected with an analyte-specific probe that is labelled directly or indirectly with a signal-generating reporter group. Frequently used labels are radioactive or non-radioactive. Enzymes also come into consideration as a label in which the enzyme catalyses a certain detection reaction. In homogeneous assays no binding to a solid phase takes place.
Intercalator compounds are often used for this which undergo a change in a measurable physical variable after intercalation in the double strand composed of analyte nucleic acid and probe such as an increase in their fluorescence (Nucleic Acids Research, 1995, 23 (5), 753-760) or a change in their fluorescence polarization. In homogeneous assays according to WO 92/02638 a probe which contains a fluorescence label as well as a fluorescence quencher is used. During the assay the label is cleaved from the quencher and the fluorescence activity is detected. The said heterogeneous and homogeneous assays require the use of chemically complicated, specially labelled probes. These are, on the one hand, complicated to prepare and, on the other hand, for each label it must be determined whether it adversely affects the hybridization efficiency.
A method for sequencing DNA is described in U.S. Pat. No. 5,453,247. In this method a set of nucleic acid fragments is produced based on the DNA whose nucleotide sequence is to be determined, wherein each set ends with one or more of the four nucleobases. All possible fragments (single stranded) of the region that is to be sequenced have to be generated. The individual fragments each differ by one nucleotide. The mass of each of these fragments is determined by mass spectrometry.
A method for the detection of nucleic acids is described in WO 95/15974 in which a labelled nucleic acid analogue probe is contacted with the nucleic acid to be detected, the nucleic acid is degraded down to the region protected by the probe and the presence of the complex is detected with the aid of the probe label.
J. Am. Chem. Soc. 1995, 117, 831-832 and Rapid Communications in Mass Spectrometry, 10 47-50 (1996) describe mass spectra of complexes composed of a peptide nucleic acid (PNA) and an oligonucleotide in which the oligonucleotide was prepared synthetically and is the same size as or larger than the region which binds to the PNA molecule by base-base interactions.
The object of the present invention was to provide a nucleic acid detection method which does not require labelled probes and which allows a parallel determination of different nucleic acids.
Therefore a subject matter of the invention is a method for detecting nucleic acids by binding a probe P to a partial sequence S contained in the nucleic acid to produce a binding product B
1
, degrading the nucleic acid to produce a binding product B
2
containing a partial nucleic acid of a defined length and detecting the binding product B
2
or a part thereof based on its mass.
A nucleic acid within the sense of the invention means all nucleic acids such as RNA and DNA known to a person skilled in the art. Double-stranded as well as single-stranded nucleic acids can be detected. In the preferred case that the probe P has strand-displacing properties, it is possible to use double-stranded nucleic acids in the method according to the invention without prior denaturation.
The nucleic acids to be detected can be degraded in a defined manner. The source of the nucleic acids to be detected is unimportant. However, a sample containing the nucleic acid to be detected is preferably subjected to a pre-treatment so that the nucleic acids are present in solution and are accessible to dissolve probes. Especially when detecting nucleic acids from cells or organisms, it has proven to be advantageous to destroy the nucleic acids by partial or complete degradation of the cell walls surrounding the nucleic acids. A variety of methods are known for this to a person skilled in the art such as digestion with the aid of proteases in the presence of detergents or/and chaotropic salts. Although it is in principle possible and even preferable within the sense of the invention to already detect nucleic acids from lysed crude samples prepared in this manner, it is, however, also possible to firstly purify the nucleic acids of interfering sample components e.g. by binding the nucleic acids to solid phases e.g. to glass-like surfaces in the presence of chaotropic salts and removing non-bound sample components. The nucleic acids bound to the solid phase can be subsequently removed again from the surface for example by low salt buffers (e.g. according to WO 95/01359).
The invention also sets no limits with regard to the function of the nucleic acids. The nucleic acids can be genomic i.e. long nucleic acids, but also nucleic acids which have already been partially reduced in size, or transcripts. The nucleic acids are particularly preferably products of a nucleic acid amplification such as the polymerase chain reaction (PCR) according to EP-B-0 201 184. In the PCR nucleic acids to be detected in the sample are amplified by contacting the sample with two primers one of which is complementary to a first strand and the other is complementary to its opposite strand on the nucleic acid to be detected. The primers are selected such that the product of an extension of the one primer can serve in turn as a template to extend the other primer using the nucleic acid to be detected as the template. The extension is carried out by a DNA polymerase using monodeoxy-ribonucleoside triphosphates. The extension products are separated between the individual extension steps from the respective templates by denaturation e.g. by a heat step. Such an amplification enables the specific amplification of a certain part of a nucleic acid even in the presence of other nucleic acids. Therefore the nucleic acids to be detected are not only naturally occurring nucleic acids but also amplified or/and modified nucleic acids.
A characteristic of the nucleic acid to be detected is the presence of a partial sequence S in the nucleic acid. This partial sequence is composed of a sequence of nucleic acid bases of a certain and previously known sequence. The partial sequence is preferably larger than 5 nucleotides, preferably between 6 and 100 nucleotides, particularly preferably between 10 and 30 nucleotides long. The partial sequence is preferably on a region of the nucleic acid that is accessible to the probe. It contains the sequence information which is intended to be the basis for the detection e.g. a sequence within which a certain mutation is presumed to occur or which is characteristic for a certain organism. This mutation can be one or several point mutations, one or several deletions, one or several insertions or polymorphisms. Mutations are particularly preferred which are an indicator for an infection by an organism, a genetic disease or a predisposition for such a disease. Examples of such mutations are deviations in the P53 gene. An example of the detection of an infection is the detection of hepatitis C virus bas

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