DE3807975C2 - Process for the optical characterization of nucleic acids and oligonucleotides - Google Patents

Description

The present invention relates to a method for optical characterization of nucleic acids and oligonucleotides.

The precise analysis of nucleic acids (single and double stranded DNA or RNA) is central to understanding life processes in the cells and in particular for determining the information content of the DNA.

Methods have already been developed for analyzing nucleic acid sequences Service. The classic sequencing method (Proc. Natl. Acad. Sci. USA 74 (1977) 5463) of nucleic acids, in particular DNA, is slow, error prone and very difficult to automate. That is why in recent years Some methods have been developed that use fluorescence staining of DNA (J. Biochem. Biophys. Meth. 13 (1986) 315, Nature 321 (1986) 674. Science 238 (1987) 336. However, none of these methods are used specific interactions between dye and nucleic acid in order to Characterize DNA. Therefore, all known methods require either several chemically different dyes (EP 02 52 683 A2, page 4; lines 38-48 and EP 02 51 786 A2; Page 3, lines 38-43, EP 02 33 053 A2) and / or several separation tracks in the separation medium (DE 36 42 939 A1). Only by the use of four chemically different fluorescent dyes four different fluorescence characteristics to characterize the Obtain nucleotides. The use of four dyes brings the following Difficulties with it: (1) It is a targeted coupling of the special Dye to the nucleotide, which is a subsequent dye coupling in makes a mixture of nucleotides impossible; (2) The different Dye scaffolds have the marked in the electrophoretic separation Nucleotide fragments result in different mobilities that are difficult can be corrected.

The use of thionucleotides for DNA sequencing inhibited exonuclease digestion is described in WO 86107612. This However, the process is poorly suited for automation.

There were intermolecular interactions between fluorescent dyes and nucleobases are investigated, but an analytical use is not possible, since there is no covalent coupling of dye and nucleobase (Chem. Abstracts 90: 163479K (1979)).  

There are also several to visualize the DNA during in-situ hybridization Methods have been developed (Proc. Natl. Acad. Sci. USA 78 (1981) 6633, Exptl. Cell. Res. 153 (1984) 61, Histochem. 85 (1986) 1). These methods are however, very time consuming or lead to unavoidable defects partially uncertain results.

It is also a method of fluorescent staining using nucleic acids enzymatic transfer of a colored thionucleotide (Nucleic Acids Res. 12 (1984) 1791). After this he can method however only a maximum of one dye molecule per DNA strand coupled to the DNA become.

The invention relates to a method for characterizing Nucleic acids and / or oligonucleotides. characterized in that one Coupling detector molecules to the nucleic acids and / or oligonucleotides on the basis of interactions, to which nucleotide the Coupling has taken place.

Oligonucleotides are understood to be those with up to 20 nucleotides contain. Molecules that consist of more than 20 nucleotides are shown here as Designated nucleic acids.

Optical characterization of the nucleic acid or oligonucleotide means that the bases of the nucleotides by coupling a fluorescent dye be identified based on optical properties and the nucleic acid or the nucleotide is detected.

Only those dyes that are suitable for characterization are suitable show different quenching capabilities, d. H. the different interactions between the dye and the four possible terminal bases. A suitable measure of this Interaction is the fluorescence lifetime of the dye.

Fluorescent dyes such as fluoresceins, rhodamines, Oxazines and especially coumarins, preferably 7-amino-4-methylcoumarin, Carbostyryles - preferably 7-amino-4-methylcarbostyryl and oxadiazoles - preferably 2- (4-biphenyl) -5-phenyl-1,3,4-oxadiazole.

This invention is particularly suitable for use in sequencing, because for the first time the information that lies in the specific chain terminations, by coupling fluorescent dyes to the terminal nucleotides is used for identification.  

The invention further relates to a method for the identification of Nucleic acids or oligonucleotides, characterized in that Thionucleotides incorporated in the nucleic acids or oligonucleotides and then specifically covalently to the sulfur of the thionucleotide Detector molecules couple. Labeling of the nucleic acid or the Oligonucleotide means that the nucleic acid or the oligonucleotide is made visible or detectable.

As detector molecules u. a. Dyes, radioactive labels, spin labels or Serve antigens. The latter can occur in an antigen-antibody reaction stained antibodies are made visible. The detection can be done in the usual way Way, e.g. B. fluorescence microscopy, spectroscopic or using the blot Technology.

The thionucleotides are suitable for incorporation into the nucleic acid or the Oligonucleotide those which have an SH- in the 2'- or 3'-position of the pentose Wear group such. B. 2 ', 3,' - dideoxy-3'-thioribose, 2 ', 3'-dideoxy-3- thioxylose, 2 ', 3'-dideoxy-2'-thioarabinose, 2'-deoxy-2'-thioarabinose, 3'- Deoxy-3'-thioxylose, 3'-deoxy-3'-thioribose. Such are particularly suitable Thionucleotides that carry the S atom on the phosphorus. d. H. the deoxynucleotide 5'-thiophosphates or nucleotide 5'-thiophosphates.

The thionucleotide is incorporated terminally and / or at any Set the nucleic acid or the oligonucleotide to chemical or preferably enzymatic way. The terminal installation takes place at Sequence analysis. The installation at any point serves to identify the Nucleic acids and oligonucleotides.

The terminal enzymatic incorporation of the thionucleotide is carried out by matrix controlled polymerization of nucleic acid with a polymerase in Presence of a varied terminator. Among the varied terminators are -α- thiophosphates or triphosphates of the above dideoxypentoses with to understand an SH group.

Enzymatic incorporation of thionucleotides anywhere DNA is preferably carried out by the enzymatic nick translation in Presence of a DNAase and a DNA polymerase. Suitable as a DNAase for example deoxyribonuclease I from bovine pancreas. As DNA polymerases come for example polymerase 1 from E. coli, the Klenow fragment and T 7- DNA polymerase into consideration.

The thionucleotides are chemically incorporated in a known manner (see Winnacker: Gene und Klone, VCH Verlagsgesellschaft, D-6940 Weinheim, 1985. Pages 44 ff). Instead of nucleotides, only thionucleotides are used used.

The dye can be bound directly to the thionucleotide. ahead setting for this, however. that the dye is an activated coupling group for  Contains sulfur. Examples of such groups are 40 α-haloacetyl, Maleimide and allyl bromide groups. Dyes of this type are, for example in the 'Bioprobes' catalog from Molecular Probes. Eugene, usa, described.

The coloring can also be done indirectly. This is done as above activated hapten described. With the help of an antigen Antibody reaction with stained antibodies or by coupling one Enzyme. like peroxidase, which triggers a color reaction, happen (cf. Proc. Natl. Acad. Scilly. USA 70 (1981) 2238, Exptl. Cell Res. 153 (1984) 61, Histochem. 85 (1986) 1).

The method according to the invention has the following advantages:

• 1. The DNA is stained immediately before the separation or the Visualization. This means that practically all of the properties of the DNA remain received for visualization. The nucleotides in the DNA are identified by the Exchange of an oxygen atom for a sulfur atom or one Hydrogen atom against an SH group did not change significantly. This clearly distinguishes the new process from the known ones Method.
• 2. The dyeing reaction proceeds quickly and under mild reaction conditions quantitatively.

The application of the present invention to DNA sequencing makes it is possible for the first time to have a separation membrane for all bases and only one dye Identification of the bases to use. This 1-lane 1-dye The concept increases the accuracy of the analysis and the Performing DNA sequencing is much easier since all Separation systems and in particular also HPLC can be used.

No wrong stops are observed in the method according to the invention, since these are not marked. Wrong stops are chain terminations that are unspecific done in the DNA and for which it is e.g. Currently there is no explanation.

The use of dideoxynucleotides additionally varied in the base enhances the resolution accuracy of the optical characterization of the terminal nucleotides. The variation in the base consists, for example, in Exchange of C for N atoms or vice versa, e.g. B. by using 3,7-diazapurine, 5-aza-7-desazapurine or 6-azacytosine.

1-lane 2-color has the same effect as the 1-lane 1-dye concept fabric concept, which also includes the spectral differentiation of the dyes in Combination with the fluorescence lifetimes.  

The new method uses fluorescence life measurement for the first time duration of dyes coupled to DNA molecules. The measurement is very sensitive, since the scattered light that otherwise interferes with steady-state measurements Excitation cannot influence the measurement. The measuring accuracy is thereby increased. In addition, the measurement of the fluorescence lifetime allows the Identification of the bases, even if their bands overlap, since the duration the lifespan is not affected by the overlap. The lifespan can for example by single shot, single photon counting or with a Streak camera can be measured when excited with a fast Flash lamp or a short laser pulse of suitable wavelength.

The sequencing method according to the invention is essentially based on the following requirements:

• 1. Matrix-controlled polymerization with a varied terminator, so that each DNA strand has a specific coupling site at the end.
• 2. Coupling of a fluorescent dye with the property. that through the different fluorescence lifetimes the terminal nucleotides be identified.
• 3. Separation of the DNA fragments on a lane using HPLC or Electrophoresis.

This process is also suitable for automation. Because currently big Efforts are being made to increase DNA sequencing accelerate, make the replacement of the radioactive marking by one Fluorescent dye, the 1-lane concept and thus made it easier Automation represents an essential step in this direction.

Another area of application for specific dye coupling to DNA represents the in-situ hybridization. In addition to the time saved by the replacement of radioactive labels by fluorescent dyes is also on the interested in the simultaneous detection of several hybrids, which by Color reactions can be realized.

The dye was used in the dyeing methods of the known sequencing methods directly to the base of the nucleotide or to the first sugar residue of the primer bound. Such modifications through additional mostly aromatic systems interfere too much with the in-situ hybridization, so that here the current methods are limited to pre-hybridizing the bases chemically through binding of haptens and through coupling sites modify to perform dye coupling after hybridization. The chemical properties are due to the drastic modifications strongly influenced.

The base modification also influences the hybridization properties very. which in changed melting temperatures and changed enzyme affinities find their expression.  

With regard to dye coupling, the invention offers the following advantages:

• 1. The base is not modified for the first time. but the phosphate or sugar rest varies.
• 2. For the first time, all bases or only a desired region can be colored become.
• 3. An increase in the dye density is possible and thus an increase in Sensitivity.
• 4. All molecules that are desired for the indirect staining reaction can be coupled.

The method according to the invention for in-situ hybridization is essentially based on the following requirements:

• 1. The nucleic acid samples can by nick translation with Thio deoxynucleotides, by matrix-controlled polymerization or by chemical synthesis.
• 2. The nucleic acid sample with any proportion of incorporated Thionucleotides are used for in situ hybridization.
• 3. Only after the hybridization is the coupling and coloring reaction carried out.

Compared to the methods above, this method is at least 4 × more sensitive since all bases and not just one base are colored here can. To quench each other's neighboring dyes too can be avoided, lipophilic solvents such as hexafluoroisopropanol can be added. Coupling reagents can be used to further increase the dye density use several dyes. The same goal can also be achieved achieve when you couple an antigen and this via antigen-antibody Detects reactions with fluorescence-labeled antibodies.

applications

In all of the examples, the dye 7-amino-N- (2- ethylaminocarbonyliodomethyl) -4-methyl-coumarin (= 4,7-IEME-Cum) used.

1. Coupling of a dye to a thiomononucleotide

13 mg (0.034 mmol) 4,7-IEME-Cum were dissolved in 625 µl dimethylformamide solved. Then 300 µl 1 mol / l buffer (Tris / HCl, pH 7.4) and 325 µl 83 mmol / l (0.027 mmol) thio-AMP in aqueous solution were added. After 1 min at 5 ° C the reaction was complete and no thio AMP more detectable.

The reaction solution was purified by column chromatography over ®QAE- Sephadex A25 with triethylammonium bicarbonate pH 7.5 (gradient elution; 20-150 mmol / l; 2 × 2 l) separated at 40 ° C. The fluorescent Function contained the product in approximately 100% yield.

The degree of purity was 80% according to HPLC analysis (reversed phase C ₁₈ silica gel with gradient elution: buffer (50 mmol / l KHPO ₄ , pH 6.5) - acetonitrile / buffer (1: 1) with the increase: 100% in 50 min). The product was eluted from the column at approximately 40%. There were no fluorescent contaminants in the product.

In a corresponding manner, the dye can be thio-TMP. Thio-GMP and thio-CMP couple and the corresponding thiodesoxynucleotides couple.

2. Identification by fluorescence lifetime

The nucleotides coupled with the dye (F) are referred to as AF, GF, CF, TF. The single shot fluorescence lifetime measurement was chosen as the measurement type.
Excitation wavelength: 383 nm
Camera filter: 435 nm
Pulse duration: less than 100 ps
Pulse number: 70

Measured fluorescence lifetimes in H ₂ O

F: 5.4 ns
AF: 6.9 ns
GF: 2.2 ns
CF: 5.5 ns
TF: 3.2 ns

3. Sequencing of DNA with 2 ', 3'-dideoxy-α-thionucleotides and specific fluorescence staining test description

The single-stranded DNA to be sequenced with a Initial part of the complementary oligonucleotide sequence to be read (Primer) hybridized. The hybrid forms the starting point of the DNA Polymerase step (b).

In the case of the matrix-controlled polymerization, starting from the hybridized oligomer (primer) a complementary counter strand to sequencing DNA template synthesized. In 4 different Reaction batches were each a thiodideoxy analog as a terminator offered in a mixture with the natural nucleotides and thus statistically distributed termination of the polymerase reaction on the growing counter chain achieved.

Labeling of DNA fragments with base-specific ends formed during sequencing can either be done by conventional radioactive labeling techniques. for example by using primer oligonucleotides labeled with ³² P at the 5 'end or by coupling fluorescent labels to the thio function of the thio-2', 3'-dideoxynucleotides after the enzymatic sequencing reaction has taken place.

Experimental Procedure

The sequence of the DNA fragment that is obtained is determined if one reads the HIV 1 pol reading frame with the restriction enzymes Bgl I and Sal 1 cuts. The fragment to be sequenced is converted into vector M13 mp 19 installed with the BamH I and Sal I interfaces and cloned.

a. Hybridization of the DNA template with a kinased Primer oligomer

One pipetted
2 µl DNA solution, 100-300 µg / ml
0.5 µl oligomer, 5 µg / ml (5'-GTAAAACGACGGCCA-3 ')
1 µl buffer: 200 mmol / l Tris / HCl pH 7.5. 100 mmol / l MgCl ₂ ,
10 mmol / l dithioerythrol, 500 mmol / l NaCl
6.5 µl water
in an Eppendorf tube and incubated the reaction mixture for 60 min at 65 ° C.

b. Sequencing reaction with DNA polymerase (Klenow fragment)

The reaction mixture from a. was cooled to room temperature.  

Then 0.8 µl (= 4 units) of DNA polymerase (Klenow- Fragment) added and the polymerization batch on a Microtiter plate divided into 4 batches of 2.5 µl each. 3 µl of each was then added to each of the reactions Given sequencing mix that starts the polymerization.

The following 4 nucleotide solutions (the numbers mean µmol / l) = "sequencing mixes" were used:

For the polymerization, the 4 batches were left at room temperature for 20 minutes stand.

c. Separation and detection

The separation and detection can be carried out using the 1-lane 1-dye principle or optionally through the 4-lane principle with radioactive labeled nucleotides. The DNA strands were cut through Electrophoresis at 75 watts in 2 h on 6% acrylamide 8 mol / l urea gels according to the method of Sanger and Coulson (FEBS-Letters 87 (1978) 107) separated. The same was obtained on detection by autoradiography Sequence as Ratner et al. (Nature 313 (1985) 277).

4. Nick translation and in situ hybridization using fluorescence detection Dye coupling to thionucleotides test description

A nick is used to incorporate the thionucleotides into the sample DNA. Translation carried out. The DNA sample (PUC 177) was used binds specifically to the centromere region of human chromosome 1. In the- situ hybridization is done with a nuclear metaphase plate, by denaturing the metaphase nuclei and the sample DNA together and are then renatured, with only the small sample DNA attached to the Chromosome 1 can hybridize. The hybridization is carried out under the Fluorescence microscope observed.  

Experimental Procedure a. Nick translation

Approach:
1 µg sample DHA (pUC 177) V = 2.5 µl, c = 435 µg / ml
Thio-d-UTP: 10 µl, c = 0.5 mmol / l
dATP / dGTP / dCTP: 5 µl each, c = 0.2 nmol / l
Nick translation buffer: 5 µl
H ₂

O: 12.5 µl

After mixing the reaction solution, 5 ul enzyme solution pipetted in, and the mixture was incubated at 15 ° C. for 90 min. To The incubation period was quickly cooled on ice and the Reaction ended by adding 50 µl stop mix.

Solutions used

• - Nick translation buffer:
  Tris / HCl (pH: 7.2): 0.5 mol / l
  MgSO ₄ : 0.1 mol / l
  Dithiothreitol: 1 × 10 ^-3 mol / l
  Bovine serum albumin (fraction V): 500 µg / ml
• - Enzyme solution:
  DNA polymerase (E. coli): 0.4 U / ml
  -DNAase I (bovine pancreas): 40 pg / µl
• - Stop mix:
  -Bromphenol blue: 0.1%
  -Dextran blue: 0.5%
  -NaCl: 0.1 mol / l
  -EDTA: 0.2 mmol / l
  -Tris / HCl (pH 7.8): 0.2 mmol / l

b. in-situ hybridization

• - preparation of the sample DNA from step a)
  Sample DNA (PUC 177): 10 µl
  Formamide: 30 µl
  20 × SSC pH 7: 5 µl
  H ₂ O: 5 µl
  Solution used:
• - 20 × SSC (PH 7.0):
  NaCl: 1.5 mol / l
  Na ₃ citrate: 0.15 mol / l
• - denaturation
  Simultaneous denaturation of the sample DNA and the cores and metaphases fixed on the slide at 70 ° C for 10 min.
• - hybridization
  overnight at 40-42 ° C.
• - detection reaction and observation
  After washing the slides, they were treated with staining solution:
• - Tris / HCl (1 mol / l) pH = 7.8: 5 µl
• H ₂ O / DMF (1: 1): 45 µl
• - 4,7-IEMeCum: 0.5 µg

Afterwards the staining solution was washed off and the metaphases of the Nuclei observed on the slide under the fluorescence microscope.

5. Selection of nucleobase-specific, erasable fluorescent dyes

The Stern-Volmer constant K _SV is a measure of the quenching of fluorescent dyes by nucleotides. Through targeted variation of the basic rhodamine structure in the JF2E to JF4E series, it is possible to produce a nucleobase-specific, erasable rhodamine. The suitability of the oxazines is also evident here (for structures, see U. Brackmann, Lambdachrome Laser Dyes, Göttingen 1994).

Claims (3)

1. Method for the detection and simultaneous identification of Nucleic acids and / or oligonucleotides to which a fluorescent dye, selected from the group of fluoresceins, rhodamines, oxazines, Coumarins, Carbostyryle and Oxadiazole, is coupled for detection, being due to different interactions between the Dye and the bases a base-specific deletion (quenching) occurs so that due to the specifically changed fluorescence life time is recognized to which nucleotide the coupling has been made. 2. The method according to claim 1 for the enzymatic sequencing of DNA, after a matrix-controlled polymerization of deoxynucleoside triphosphates and varied terminators with a polymerase resulting fragments before or after electrophoresis Fluorescent dyes, selected from the group of fluoresceins, Rhodamine, Oxazine, Coumarine, Carbostyryle and Oxadiazole and are characterized by their fluorescence lifetime. 3. The method according to claim 1 for detection and simultaneous identification of nucleic acids and / or oligonucleotides with the help of coupled Dyes, with the addition of spectral differentiation Dyes are used in combination with the fluorescence lifetimes.