JP2002533097A - Detection of specific nucleic acid sequences by polymerase nucleotide incorporation - Google Patents

Abstract

  (57) [Summary] A rapid and effective method of detecting a target DNA or RNA sequence is provided. A primer having a 3'-hydroxyl group at one end and a nucleotide sequence sufficiently homologous to the nucleotide identification sequence in the target DNA is selected. The primer is hybridized to the identified nucleotide sequence on the target DNA or RNA sequence, and the reporter molecule is synthesized by gradually attaching complementary nucleotides to the primer. The complementary nucleotides here comprise nucleotides labeled with a fluorophore. The fluorescence emitted by the fluorophore on a single reporter molecule is detected to identify the target DNA or RNA sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[Related Application] This application is a US provisional application SN 60/11 filed on December 18, 1999.
Claim 3,139 benefits.

STATEMENT REGARDING FEDERAL RIGHTS This invention is subject to United States Department of Energy, Contract No. W-7405-ENG-36.
Made with government assistance under The government has certain rights in the invention.

[0003] The present invention relates generally to the detection of nucleic acid sequences, and more particularly, to the selective mixing of fluorescent markers for detecting nucleic acid sequences.

BACKGROUND OF THE INVENTION [0002] The rapid and effective detection of specific nucleic acid sequences in biological samples is in a variety of fields including molecular biology, biotechnology, immunology, medical diagnostics, forensic analysis and food quality control. Plays a central role in One of the most commonly used techniques for detecting a particular nucleic acid sequence is a Southern blot. This is a hybridization technique in which the fragments to be investigated are separated by size by gel electrophoresis and transferred from the gel to a nylon nitrocellulose filter. The radioactive probe is then added to the filter for performing the hybridization. After washing away excess probe, the band containing the target nucleic acid is detected by exposing the X-ray film to the filter.

[0005] Despite its generality, Southern blotting suffers from several limitations. It involves a series of manual, intensive approaches that cannot be performed unattended and that cannot be easily automated. The method of separating fragments by gel electrophoresis and then detecting bands by autoradiography is a time-consuming task that tends to have poor quantitative accuracy and low reproducibility.

[0006] The use of radioactive probes poses a set of safety and environmental concerns. Lack of adequate sensitivity is another limitation, which is the polymerase chain reaction (PCR).
And the development of related target amplification methods. PCR consists of the selective amplification of a target DNA sequence in a sample. Amplification products are usually detected by a dye that stains the nucleic acid, or by hybridization with a sequence-specific probe. Amplification methods, however, can induce ambiguity resulting from variability in contamination or amplification capacity. Therefore, there is a need for powerful analytical methods that provide accurate quantification and molecular weight assessment for target DNA or RNA.

[0007] Various objects, advantages and novel features of the present invention are set forth in part in the description which follows, and in part will become apparent to those skilled in the art in the following tests, or as determined by the practice of the invention. Is also good. The objects and advantages of the invention may be realized and attained by means of the instruments and combinations particularly pointed out in the appended claims.

SUMMARY OF THE INVENTION To achieve the above and other objects, the present invention, as embodied and broadly described herein, identifies a target DNA or RNA sequence based on the object of the present invention. Includes a method for A primer having a 3'-hydroxyl group at one end and having a nucleotide sequence sufficiently homologous to hybridize to the identified nucleotide sequence in the target DNA or RNA is selected. The primer is hybridized to the identifying sequence of nucleotides and a reporter molecule is synthesized on the target sequence by extension of the primer by progressively binding the complementary nucleotide to a primer complementary to the corresponding nucleotide of the DNA or RNA sequence. At this time, the complementary nucleotide includes a nucleotide labeled with a fluorophore. The fluorescence emitted by the fluorophore on each reporter molecule is detected to identify the target DNA or RNA sequence.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, a novel method allows for the direct detection of a specific nucleic acid sequence in a biological sample. The basis of the research method is to observe the presence of specific nucleic acid sequences from bacteria, humans, plants or other sources. A nucleic acid sequence may be a DNA or RNA sequence and may have characteristics of a particular taxon, a particular physiology, or a particular genetic characteristic.

The method consists of synthesizing a fluorescent nucleic acid reporter molecule in vitro using a relatively short sequence of the target, a template similar to that shown in FIGS. 1A-1E. DN
The A target (FIG. 1A) is denatured according to well-known methods to form a single-stranded DNA target (FIG. 1B). A short oligonucleotide primer that is specific and complementary to the target then hybridizes to the single-stranded DNA target. A suitable polymerase and free nucleotides are added to the sample. One of these oligonucleotides is at least partially labeled with a fluorophore. If the target is present in the sample, the primer binds to the identifying sequence of the target (FIG. 1C) and the polymerase incorporates labeled and unlabeled nucleotides (FIG. 1D).
, Reconstruct the complement of the target as shown in FIG. 1E. If the concentration of labeled nucleotides is kept below the concentration of unlabeled nucleotides, most of the labeled nucleotides will be incorporated into the reporter DNA molecule. Nevertheless, some free (ie, unbound) labeled nucleotide remains in the reaction mixture, but the fluorescence from each synthetic reporter molecule is much more than the fluorescence of the free nucleotide background during the single molecule detection time. Will be strong.

As is well known and described in the art, a sample is analyzed in a single molecule detector. Detection of a synthetic reporter molecule indicates the presence of the target sought. Since the reaction proceeds until the reporter molecule is 100 or 1000 bases in length, the fluorescent signal from the reporter molecule is much stronger than the background fluorescence from the free labeled nucleotide. The novel methods described herein combine the advantages of flow-based analytical systems (system automation, speed, reproducibility) with the outstanding sensitivity of single molecule detection. The sensitivity of this method allows for the direct detection of specific genes without having to use amplification methods such as PCR, and represents an advantage over recent methodologies in terms of sensitivity, speed, and cost per analysis. The non-radioactive approaches described herein for ultrasensitive detection of specific sequences have been applied in a wide variety of fields, such as gene identification, gene mapping, medical diagnostics and biotechnology.

EXAMPLES As examples, experiments were performed on the detection of pUC19 DNA (2686 base pair plasmid). Prior to all experiments, pUC19 DNA was converted to 1568 bp in length.
And the restriction endonuclease Bg resulting in two fragments of 1118 bp
Digested with II. As a control, the same experiment was performed, except that pUC19 DNA was replaced with lambda DNA. A specific sequence of the 1568 bp pUC19 fragment was detected with a sensitivity of a single molecule level. The lambda DNA control gave a negative result.

A Design of primers. Primer sequences should be specific to the target being sought. Primers are generally 15-30 nucleotides in length. Primer lengths longer than 15 nucleotides ensure that they will not specifically anneal to non-target nucleic acids. Generally, a primer sequence has the following properties. 1 Not an internal secondary structure that interferes with hybridization and extension. 2 Average distribution of high G / C and A / T domains (45-55%). 3 For the example, we used the following 24-mer primers that anneal to nucleotides 352-375 of pUC19. 5'-d (CGC-CAG-GGT-TTT-CCC-AGT-CAC-GA
C) -3 '(SEQ ID NO: 1)

B Extraction and isolation of nucleic acids. Common extraction methods, such as phenol extraction, can be used for DNA isolation from the sample under test. See, for example, Reference 1 for nucleic acid extraction protocols.

C Reporter synthesis. i reagent

[0016]

[Table 1]

Ii. Denaturation of target DNA for 5 minutes at 95 ° C. iii. Slow and complete mixing of all reagents. Finally add the enzyme. Centrifuge briefly to collect the sample at the bottom of the tube.

Iv extension: incubate at 72 ° C. for 3 hours. The appropriate temperature is the dNTP for the primer to extend along the target DNA molecule.
Selected for hybridization. If the temperature is too low, non-specific annealing will increase. The optimal hybridization temperature is determined by The Whitehead Institute for Biochemical Research (The Whi
head Institute for Biomedical Rese
arch), which can be used for a given software routine, such as PRIMER, for a given primer / target pair. For this example, the optimal temperature for Taq DNA polymerase activity is 72 ° C.

V Optional: Addition of a "stop" solution to terminate enzyme activity. If the reaction did not stop and the target reached a stable size, the amount of dye incorporated, ie, the reporter fluorescence intensity, would be proportional to the size of the fragment.

Vi Optional: Free, unincorporated labeled nucleotides can be removed by physical means (
For example, removal by precipitation, filtration, chromatography). In the exemplary results reported herein, a large fraction of unincorporated labeled nucleotides was
Quick Nucleotide Removal Kit (Quagen)
, Valencia, CA) according to the product protocol. Conversely, the primer can be labeled with a suitable anchoring group (eg, biotin) that allows isolation of the reporter by physical means (eg, a solid support, magnetic beads).

D Analysis by Single Molecule Detection References 2 and 3 or US Pat.
Single molecule detectors such as those described in 209,834 are used for fluorescence detection from reporter molecules. Suitable flow cytometer devices and methods for single molecule detection are described in US Pat. No. 5,558,998, published Sep. 24, 1996, and filed Oct. 9, 1998, which are incorporated by reference. See U.S. Patent Application Serial No. 09 / 169,025. Depending on reaction conditions such as initial nucleotide concentration and temperature, it may or may not be necessary to remove unincorporated labeled nucleotides as described in the Procedures section. In this example, the reaction mixture was diluted 1000-fold to 50 ml. This dilution results in unincorporated nucleotide concentrations in the nanomolar range and reporter concentrations in the picomolar range. Thus, when a sample is analyzed by single-molecule detection, unincorporated nucleotides produce a constant background signal, and reporters containing hundreds of labels produce a single fluorescent burst with a stronger amplitude than that of the background. FIG. 2 shows the experimental results for the detection of pUC19 in this example. FIG. 3 shows a control experiment using lambda DNA as a target.

If the reporter synthesis reaction proceeds to completion, the amount of labeled nucleotide incorporated will be the same for the same target. Thus, each single molecule burst will show the same amplitude as shown in the simulation of FIG.

E Analysis by Single Molecule Electrophoresis Another method to avoid detection hindered by free nucleotides is described in ref. 3 and US Pat. No. 5,209,834, which is incorporated by reference. Perform "single molecule electrophoresis" as described. In this method, a fluorescently labeled molecule (freely labeled nucleotide and in this case a reporter molecule)
Is determined by single molecule sensitivity. Single nucleotides exhibit electrophoretic mobilities that are very different from the mobilities of the nucleic acid targets, eliminating interference from free nucleotides. Therefore, each single molecule burst shows the same amplitude as shown in the simulation of FIG. A bar graph of the amplitude of the burst will indicate the size of the target to be searched. This method also allowed the size of the target to be determined, even if the reaction was not completed.

References (incorporated herein by reference) 1 “DNA Probes”, G. Keller and M. Mannack, Stockton Press, New York, 1993, Chapter 2. 2 "Single-molecule detection of specific nucleic acid sequences in unamplified gene DNA", Ekastro and JK Williams, Anal. Chem. 69, 3915-3920 (1997). 3. "Single-molecule electrophoresis," Ekastro and EB Schera, Anal
.Chem. 67, 3138 (1995)

The foregoing description of the present invention has been set forth in an illustrative and descriptive sense, and is not intended to be exhaustive or to limit the invention to the precise disclosure. . And obviously many modifications and variations are possible in light of the above teaching.

Embodiments have been chosen and described in order to best explain the principles of the invention. Its implementation will enable the present invention to be most suitably utilized in various embodiments and with various modifications to suit the particular application contemplated by those skilled in the art. It is meant that the scope of the invention is defined by the claims appended hereto.

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic view illustrating a process of the present invention.

FIG. 2 is a graph showing experimental results for detecting specific sequences of pUC19 DNA at a single molecule sensitivity level according to one embodiment of the present invention.

FIG. 3 is a graph showing results for a control experiment performed under the same identification conditions as those corresponding to the experimental results shown in FIG. 2, except that the target was replaced with lambda DNA.

FIG. 4 is a graph showing a simulation of a single molecule fluorescent signal from a reporter molecule according to a second embodiment of the present invention.

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Claims (12)

[Claims] 1. A method for identifying a target DNA or RNA sequence, comprising: a primer having a 3'-hydroxyl group at one end and having a nucleotide sequence sufficiently homologous to hybridize with an identification sequence of a nucleotide in the target DNA. Selecting a primer, hybridizing the primer to an identifying nucleotide sequence of the target DNA or RNA sequence, by gradually binding nucleotides to a primer that is complementary to the corresponding nucleotide on the target sequence to form a reporter molecule. Extending the primers along the target sequence, wherein the complementary nucleotides comprise nucleotides labeled with a fluorophore, and fluorescence on individual reporter molecules to identify the target DNA or RNA sequence Fluorescence emitted by the group Method comprising the steps of. 2. The method of claim 1, wherein the primers are at least about 15 nucleotides to specifically hybridize to an identifying sequence of nucleotides for the target DNA or RNA. 3. The method of claim 1, wherein the one or more nucleotide types are at least partially labeled with a fluorescent label.
Forming a mixture of TP, dGTP, dCTP, and dUTP nucleotides; denaturing the target DNA or RNA; adding a polymerase effective to catalyze the synthesis of a reporter molecule from the mixture of nucleotides; and Incubating the mixture, the target DNA or RNA, and the polymerase for a time effective to extend the primers to the desired length. 4. The method according to claim 3, wherein the nucleotide concentration in the mixture of nucleotides having a fluorescent label is lower than the nucleotide concentration without the fluorescent label. 5. The method of claim 1, further comprising the step of separating free, non-incorporated nucleotides after the completion of the coupling reaction. 6. The method of claim 5, wherein the primers are at least about 15 nucleotides to specifically hybridize to an identifying sequence of nucleotides for the target DNA or RNA. 7. The method of claim 5, wherein the one or more nucleotide types are at least partially labeled with a fluorescent label.
Forming a mixture of TP, dGTP, dCTP, and dUTP nucleotides; denaturing the target DNA or RNA; adding a polymerase effective to catalyze the synthesis of a reporter molecule from the mixture of nucleotides; and Incubating the mixture, the target DNA or RNA, and the polymerase for a time effective to extend the primers to the desired length. 8. The method according to claim 7, wherein the nucleotide concentration in the mixture of nucleotides having a fluorescent label is lower than the nucleotide concentration without the fluorescent label. 9. The method of claim 1, further comprising the step of separating free, non-incorporated nucleotides from the reporter molecule by single molecule electrophoresis. 10. The method of claim 9, wherein the primers are at least about 15 nucleotides to specifically hybridize to an identifying sequence of nucleotides for the target DNA or RNA. 11. The method of claim 9, wherein the one or more nucleotide types are at least partially labeled with a fluorescent label.
Forming a mixture of TP, dGTP, dCTP, and dUTP nucleotides; denaturing the target DNA or RNA; adding a polymerase effective to catalyze the synthesis of a reporter molecule from the mixture of nucleotides; and Incubating the mixture, the target DNA or RNA, and the polymerase for a time effective to extend the primers to the desired length. 12. The method according to claim 11, wherein the nucleotide concentration in the mixture of nucleotides having a fluorescent label is lower than the nucleotide concentration without the fluorescent label.