Method for generating multiple double stranded nucleic acids

Details Method for generating multiple double stranded nucleic acids Method for generating multiple double stranded nucleic acids

1998-05-22

2001-12-04

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091510, C536S026740, C536S025320, C536S025400

Reexamination Certificate

active

06326143

ABSTRACT:

BACKGROUND OF THE INVENTION
The determination of analytes in samples plays an important role in either environmental or human diagnostics analysis. The infection or pollution of samples by substances coming from the environment is becoming a major focus of the industry. Because many of the substances are present in samples in very low amounts, the methods for the analysis must be very sensitive. This is especially true for immunological determinations or analyses based on the occurrence of nucleic acids.
The first increase in sensitivity to be achieved in nucleic acid assays was realised by the possibility to amplify the amount of analyte nucleic acid in a sample. This has made possible the determination of even very low amounts of nucleic acids present in a sample. One example of a method for the amplification of analyte nucleic acids in a sample is the so-called polymerase chain reaction which is described in detail in U.S. Pat. No. 4,683,202. This method uses two primers chosen such that the sequence of the first primer is complementary to a region of the target nucleic acid to be amplified and the sequence of the second primer is homologous to a sequence on the target nucleic acid such that the elongation product of one primer can be used as a template for the elongation of the other primer. This method yields multiple copies of the nucleic acid to be determined.
As a further development of this method in EP-A 0 379 369 there is described a method for converting an analyte polynucleotide into a polynucleotide having at one terminus a nucleobase sequence which is complementary to the sequence at the other terminus. This newly constructed polynucleotide containing a fragment of the analyte nucleic acid is capable of being amplified using only one primer sequence. This procedure, however, has the disadvantage that the termini of the nucleic acid produced can hybridise to each other and therefore can prevent annealing of the primer. Therefore the amplification is not very effective. Further, the preparation of the amplifyable intermediate product requires the use of two different primer sequences and hence knowledge of two different sequences in the nucleic acid to be determined or amplified.
Therefore, it is the object of the present invention to provide the art with a method for generating multiple double stranded nucleic acids which can be used to determine analytes, where only one primer sequence is used.
SUMMARY OF THE INVENTION
Subject of the present invention is therefore a method for generating multiple double stranded nucleic acids by
a. elongating a primer molecule comprising a nucleobase sequence B′ by using one or more nucleotide(s) and a target nucleic acid T that acts as a template for the elongation of said primer such that the elongation product E formed is capable of acting as a template for the elongation of a further primer molecule containing the nucleobase sequence B′;
b. separating the target nucleic acid T from said elongation product E,
c. using said elongation product E as a template for the elongation of a further primer molecule yielding an elongation product E′,
d. repeating the steps of elongating primer molecules and of separating said elongation products a sufficient number of times to achieve the desired amount of double stranded nucleic acid.
A further subject of the invention is a method for the determination of an analyte using this method, especially
binding to said analyte a target nucleic acid T having a region comprising an analyte-specific region A and a region comprising an analyte-nonspecific nucleobase containing sequence B;
hybridising to said target nucleic acid a primer comprising a nucleobase sequence B′ complementary to said sequence B;
elongating said primer using said target nucleic acid as a template to form a first elongation product E by the covalent attachment of one or more nucleotides to said primer;
determining the occurrence of said elongation product as a measure of the presence or amount of the analyte.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, a method for generating multiple double stranded nucleic acids is a method for the creation of a large number of identical double-stranded nucleic acids. This method will comprise, but not necessarily essentially consist of, an amplification of these double stranded nucleic acids. The amplification may be linear, but may preferably be perfectly or imperfectly exponential. Perfectly exponential would be an amplification wherein the number of nucleic acids created in n amplification steps amounts to 2
n
, whereas in an imperfect exponential amplification this theoretical amplification factor will not be achieved. The double stranded nucleic acids generated can be subject to further processing steps like chemical modification or physical treatment, like strand separation yielding single stranded nucleic acids.
A primer according to the present invention is a molecule comprising a nucleobase sequence B′ which has an affinity to a nucleobase sequence B contained within a target nucleic acid and is capable to be elongated by nucleotide(s) at an elongatable end. Preferably, the nucleobase sequence B′ of the primer is substantially complementary to a nucleobase sequence B in the target nucleic acid. Preferably, the primer has such a specificity for the target nucleic acid or the nucleobase sequence B such that it does not hybridise under the conditions chosen to nucleic acids not intended to be used as target for generating multiple double stranded nucleic acids. The primer according to the present invention is preferably a nucleic acid, for example DNA or RNA, DNA being preferred. The primer can act as substrate for attachment of one or more nucleotides The primer has a 3′-terminal hydroxyl group to which mononucleotides can be attached enzymatically using the protruding end of the target nucleic acid as a template, thus forming an elongation product of the primer. In the following, the end of the primer intended to be elongated is named elongatable end of the primer. Primers are generally used in large excess over the target nucleic acid.
In a special embodiment the primer contains a stretch of at least 10 nt of continously connected nucleotides bearing the same nucleobase, preferably a pyrimidine nucleobase like T or C, most preferred cytosine. Most preferably, this stretch is located within sequence B′. At the elongatable end of the primer the primer may contain one or more nucleotides which differs from the nucleotides in the above mentioned stretch and improves the selectivity of a the binding of the primer to the nucleobase sequence B of the target nucleic acid T. In a preferred embodiment the primer contains a stretch of identical bases of preferably at least ten identical bases in consecutive order and, in the direction of the elongation, three or less bases differing from the before mentioned bases. Preferably only two or one such base is attached to this terminus of the stretch. Preferred bases at the elongatable terminus of the primer are bases which can form base pairing with the before mentioned stretch of identical bases in the primer. However, the number of these bases is less than the number useful to produce stable intra-probe structures. The primer can have attached further moieties, provided that they do not make impossible the hybridisation of the primer to the target and subsequent elongation. Especially, moieties possibly attached to the primer comprise labelling groups. The sequence B′ is predetermined and chosen such that it fits to the corresponding stretch of nucleobases in the target nucleic acid.
A nucleobase sequence according to the present invention is composed of naturally or non-naturally occurring nucleobases linked together by a backbone, for example, a sugar-phosphate backbone as in usual nucleic acids. The nucleobase sequence, for example, determines the specificity with which a primer binds to a target nucleic acid. For achieving a certain specificity it may be

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