DNA detection by means of a strand reassociation complex

Details DNA detection by means of a strand reassociation complex DNA detection by means of a strand reassociation complex

1999-06-04

2002-06-25

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S912000, C435S210000, C536S024300, C536S024330

Reexamination Certificate

active

06410235

ABSTRACT:

The invention concerns a method for the detection of nucleic acids using the amplification of the nucleic acid with the aid of labelled primers and detection of the amplificate with the aid of a capture probe.
Biospecific binding assays which enable the detection of certain analytes or analyte characteristics by means of molecular recognition mechanisms have become indispensable in diagnostics and bioanalytics. In this connection hybridization assays have become firmly established in recent years in addition to immunoassays and receptor ligand assays. Hybridization assays utilize the principle of nucleobase pairing (A::T; G:::C) for the molecular recognition of certain analyte nucleic acids (e.g. DNA, RNA) by probes with the desired specificity. Thus for example oligonucleotide probes which are composed of 18-20 nucleotides in a chosen sequence enable unequivocal detection even over the entire human genome.
Hybridization assays (=probe based assays) have been given an interesting and promising extension by so-called NA chip technologies. In these at least 2 and usually several to very many probes with different sequences and thus different specificity are bound in a geometric pattern in separate areas on a test carrier so that a corresponding number of hybridization reactions between the probes and nucleic acid analyte segments or different nucleic acid analytes can be carried out concurrently. Under suitable reaction conditions e.g. sequence selection, buffer environment, salt content and above all the incubation and wash temperature, it is possible to keep only those hybridization complexes bound to the solid phase in which all the nucleotides contained in the oligonucleotide probe are complementary to the corresponding nucleotides in the analyte molecule resulting in the full binding strength. This is then referred to as complete base pairing (perfect match, PM).
Hybridization complexes which contain mismatches (MM) are detached under such conditions. Under optimal conditions it is even possible to unequivocally distinguish between complexes with complete base pairings and complexes with 1-point mismatches (single base transitions). Since this occurs concurrently on the solid phase when using a geometric pattern of capture probes (array), it is referred to as probe array testing.
The capture probes can all have a constant length (number of nucleotide building blocks) or the oligo length can be inversely proportionally matched to the GC content. In the first case a common melting temperature Tm can be achieved for all completely paired hybridization complexes by buffer additives which for example strengthen AT bonds to such an extent that the Tm is independent of the nucleobase sequence and is only dependent on the oligo length. Examples of such additives are tetramethylammonium chloride (TMAC) and tetraethylammonium bromide. In the second case the stated length adaptation results in a Tm levelling. The capture probes can have chemically different backbones which carry the specificity-mediating nucleobases e.g. deoxyribosyl-phosphodiester strands (=>DNA), ribosylphosphodiester strands (=>RNA) or they can belong to a non-natural class of substances e.g. N-(2-aminoethyl)glycyl or N-(2-aminoethyl)glutamyl strands (=>PNA, WO 92/20702).
Probe array testing is of interest for many molecular biological or diagnostic applications. These include multipathogen testing (simultaneous detection of different pathogens on a gene level), (sub)typing of organisms, analysis of genetic diversity (polymorphisms, mutations), sequencing of genes or genomes etc.
Nucleic acids are relatively complex analytes which usually have to be firstly isolated, then amplified and, in the case of DNA, rendered single-stranded (denatured) before they can be used in a probe based assay or probe array testing. This processing and the fact that complementary nucleobases also have a tendency for base pairing within one and the same strand result in some typical difficulties such as a variable analyte titre in the reaction solution due to variations in the efficiency of the isolation or amplification, a suboptimal denaturing efficiency, reassociation of the single strands of a DNA to form the original double strand which competes with the hybridization of a single strand with a probe, internal strand hybridization (formation of secondary structures e.g. hairpin loops or cross formations) which compete with the probe hybridization. This becomes more pronounced as the palindrome index increases i.e. the degree of self-complementarity of a DNA or RNA strand.
Especially the last two phenomena essentially determine the accessibility of the sequence region of the analyte which is the basis for the test and hence limit the overall performance of an entire array of capture probes.
The so-called PCR method (polymerase chain reaction, U.S. Pat. No. 4,683,202) is usually used to amplify the analyte nucleic acid. In this method it is possible to already incorporate a detectable group during the amplification e.g. a digoxigenin derivative (DIG-labelling, EP-B-0 324 474). This can be achieved by replacing a part of the dTTP by DIG-dUTP in the nucleoside triphosphate mixture.
A method is described in DE-A-3807994 (U.S. Pat. No. 5,476,769) in which detectably-labelled amplicon strands are hybridized to an immobilizable capture probe and the hybrids that are formed are detected.
The so-called sandwich hybridization method is described in EP-A-0 079 139 in which a nucleic acid to be detected is detected by hybridization with a capture probe and a detection probe which are complementary to different regions of the nucleic acid.
The object of the present invention was to improve hybridization assays which are based on a capture reaction of amplificates that have been generated while incorporating a label, in particular with regard to their discrimination between two or more nucleic acids with very closely related sequences.
A subject matter of the present invention is a method for the selective detection of a nucleic acid comprising the steps amplification of the nucleic acid or of a part thereof with the aid of two primers one of which can hybridize with one strand of the nucleic acid to be detected and the other can hybridize with a complementary strand thereto at least one of which contains a bound detectable label and to form each time an extension product of these primers in a reaction mixture, binding a capture probe to one of these extension products to form a hybridization complex which contains the capture probe and at least this extension product, separating the extension product bound to the capture probe from non-bound components of the reaction mixture and determining the detectable label bound to the capture probe wherein the capture probe is selected such that it can bind with the strand of the extension product which can also hybridize with the extension product formed by extension of the labelled primer.
The nomenclature of the individual components of the amplification as defined by the invention are shown schematically in FIG.
1
. The meanings are as follows:
P1 forward primer
P2 reverse primer
T1 sense daughter strand (sense extension product)
T2 anti-sense daughter strand (anti-sense extension product)
M1 anti-sense template strand (template); strand of the nucleic acid to be detected
M2 sense template strand (template); opposite strand to the nucleic acid to be detected
A complex that is already bound to the solid phase comprising the strands T1 (unlabelled), T2 (5′-terminally labelled) and the solid phase-bound probe P is shown schematically in FIG.
2
. The rectangle is a detectable label.
The position of the regions with mutations in the segment is shown in FIG.
3
.
A folding proposal for the rpo &bgr;-gene segment is shown in FIG.
4
.
FIG. 5
shows a comparison between the ideal situation (rod-shaped DNA) and the real situation A (folded). It is clear that the accessibility of the terminal label is in fact greatly impaired. This impairment is reduced or avoided by

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