DE4011991A1 - Simultaneous sequencing of several DNA samples - by cloning into separate vectors, complementary strand synthesis from specific fluorescent labelled primers, electrophoretic sepn. etc. - Google Patents

Abstract

Method for DNA base sequencing comprises, (1) cloning a no. of DNA samples in different vectors (1-4), (2) synthesising the complementary strands of the samples (5-8) in admixture with primers (9-12) each primer being able to hybridise with only one particular vector and each labelled with different fluorophores having different emission wavelengths, (3) prepn. of 4 DNA-fragment gp. each with different end gps. from the resulting mixts. (i.e. 5-9, 6-10, 7-11, 8-12) and (4) electrophoretic migration of the fragment gps. and determination of fluorescence emission from the fragments in each gp. USE/ADVANTAGE - This method does not require complex sample pretreatments and, unlike the conventional multiplex DNA sequencing procedure, is not limited to samples which can be labelled with radiosotopes. Many samples can be processed simultaneously and no correction is required for differences in electrophoretic migration rates caused by different labelling dyes.

Description

The invention relates to a method for effective base sequence determination.

A conventional technique used for the determination of DNA base sequences is used is autoradiography. On the other hand, there is a real-time after recently white system using fluorescent markers. At This technique uses DNA fragments with four different fluorophores corresponding to the four respective types or types of connection types, or End groups or terminal bases marked and on a hiking trail or Migration path and then injected to walk a certain distance a laser beam is applied to it to observe fluorescent light to be observed emit. The end groups of the DNA fragments can by the wavelengths of the fluorescent light can be recognized and based on the detection time, the DNA Base sequence can be determined.

Another technique uses DNA fragments that are four varieties of Have end groups and are labeled with a single fluorophore, on four different migration paths are injected in order to emit the fluorescent light measure and determine the DNA base sequences.

The conventional techniques mentioned above have the deficiency that for each complicated preparations have to be made.  

To avoid these complicated preparations, a method for DNA base sequence determination, which includes

• (1) a step 1, in where different DNA samples are inserted into different vectors or be incorporated or incorporated;
• (2) a step 2 in which in a single Test tubes simultaneously complementary chains to the DNA samples be synthesized to form DNA fragments;
• (3) a step 3 in which the DNA fragments are separated electrophoretically and immobilized on filters;
• (4) a step 4 in which a DNA probe labeled with a radioisotope is attached one of the DNA samples with which they are the only one to hybridize can to the DNA fragment for determining the DNA base sequence in the Detect DNA sample;
• (5) a step 5 in which the radioisotope labeled DNA probe is washed out and another with a radioisotope labeled probe is attached to another DNA sample with which it can hybridize to the DNA fragment to determine the DNA base sequence detect the DNA sample and,
• (6) a step 6 in which the procedure according to Step 5 is repeated for the other remaining DNA samples until theirs Base sequence can be determined.

The above-mentioned method allows the simultaneous reaction and electrophoresis to determine the DNA base sequences of many samples. This technique was developed by G.M. Church et al. "Multiplex DNA sequencing", Science, Vol. 240 (1988), pages 185 to 188.

The above method cannot be used for samples that are different are marked as the radioisotopes because of the DNA band pattern on the filter must be fixed. The reason is that the use of fluorescent probes results in light scattering through filters, resulting in sensitivity that is too low results.

It is the aim of the present invention to provide a method which the Avoids difficulties of the above-mentioned conventional technique and one DNA base sequence determination by fluorescence light detection with a variety of samples to be processed at the same time.  

As a result of the inventors' investigations, it has now been found that the above-mentioned aim of the invention can be achieved with a method, that includes: a step 1 in which different DNA samples in each different vectors are cloned, a step 2 in which different primers are produced, which are hybridized only with the corresponding vectors can and which are labeled with different fluorophores; one Step 3, in addition to the samples obtained in Step 1, mixed with the above mentioned various primers, complementary chains can be synthesized; a step 4 in which from the known mixtures obtained in step 3 were, four DNA fragment groups, each with four different end groups were manufactured; and a step 5 in which the end groups classified four DNA fragment groups on individually different ones Electrophoretic webs are allowed to migrate electrophoretically and that of the DNA fragments from each DNA fragment group emitted fluorescent light is pointed.

Based on the new knowledge, the inventors have the present one Invention further studied and completed. A method according to the invention for DNA base sequence determination is as follows:

Single-stranded DNA samples are produced in which a large number of Base sequences, each inserted into different vectors, can be determined should. Primers, the number of which correlates with the number of types of vectors dated whose base sequences are different from each other and which only with a particular vector can be hybridized with respective Fluorophores marked with different emission wavelengths. The one with the marked respective fluorophores with different emission wavelengths Primers come with the multitude of corresponding different vectors for the respective single-stranded DNA samples hybridized in such a way that all DNA samples mixed and implemented with all primers; the mixture of the reacted Samples are divided into four parts; in the mixtures of the four parts Samples are synthesized individually complementary chains, so that the end groups of which are adenine, cytosine, guanine or thymine, whereby individually a DNA Fragment group is produced in which the end group of the DNA fragment adenine is a DNA fragment group in which the end group of the DNA fragment is cytosine is a DNA fragment group in which the end group of the DNA fragment guanine  and a DNA fragment group in which the end group of the DNA fragment Is thymine; and the respective DNA fragment groups become electrophoretic let wander; individual fluorescent light from the DNA fragments each group is emitted is detected and the samples are separated and according to the different emission wavelengths of the plurality of DNA samples detected.

As vectors can be used e.g. B., a well-known M13 vector, YAC Vector, pUC vector etc. The single-stranded DNA samples can be of one type and Way are made, e.g. B. that the desired or target DNA with a certain part of the vector is cloned and then alkali-denatured, such as known. Each vector has a large number of cloning parts. Anyone of the primers can can be obtained on the way that the oglionucletide with 18 to 20 bases, which is complementary to a part of the vector, artificially synthesized and is fluorescence-labeled. Since the primers obtained in their sequence configuration are different from each other, you can only use a special one individual vectors can be hybridized. The fluorophores that are the primers can mark FITC (fluorescein isothiocyanate) with 515 nm maximum Emission wavelength, NBD-F (4-fluoro-7-nitrobenzofurazan) with 540 nm maximum Emission wavelength, TRITC (tetramethylrhodamine isothiocyanate) with 575 nm maximum emission wavelength, Texas red with 605 nm maximum emission wavelength, etc.

The procedure for hybridization of the primers with the vectors and the formation of the DNA fragments by synthesis of complementary chains can be used with usual Methods are carried out.

The base sequences of a variety of single-stranded DNA samples with each different DNA base sequences can e.g. B. thereby determined at the same time that the DNA fragment groups according to the variety of the end groups are classified to be hiked on their respective hiking trails and that thereafter the fluorescent light that is emitted by applying a Laser beam on the sections with a certain distance to which the DNA fragments migrate through known wavelength separation devices, so like rotating filters or a prism are separated and each of the separated Wavelengths individually with a two-dimensional fluorescent light detector  is detected, whereby the samples are separated and detected, and on the other hand, the type of end groups of each DNA fragment is known because it is known on which migration path the fluorescence is emitted.

The trails can be on a polyacrylamide gel or agarose gel between gel support plates, such as. B. quartz or glass plates are formed.

In brief, the method of DNA base sequence determination according to the invention can be summarized as follows. Different primers are used with Fluoropho marked with different emission wavelengths. To samples from different DNA fragments that are hybridized with the primers Complementary chains synthesized in the mixed state for each end group. The complementary-chain-synthesized mixtures are based on different Walk hiking trails according to the respective types of end groups calmly. Fluorescent light emitted from sections to which it is directed have migrated and on which a laser beam is applied, is detected and the passing DNA fragments are detected. The types of sample and the end groups are determined by the emission wavelength and the Location of the migration pathways, resulting in the DNA base sequence of each sample is determined.

Further advantages, features and possible uses of the present Invention result from the following description in connection with the Drawing.

Figure 1 is a sample sectioning diagram of an embodiment of the invention.

FIG. 2 is an illustration showing groups of samples made in the steps of FIG. 1.

3 is a schematic view illustrating a multicolor fluorescence detection type electrophoresis device used in an embodiment of the present invention, and FIG

Fig. 4 is an enlarged view of line images displayed on a screen of the multicolor fluorescence detection type electrophoresis device used in an embodiment of the present invention.

An embodiment of the invention is described below with reference to FIGS. 1 to 4. First, in the embodiment, two double-stranded DNAs (DNAa and DNAb) are isolated, the base sequences of which are to be determined. In the present embodiment, a front and a back base sequence of the two double-stranded DNAs is determined in the right and reverse directions, ie a total of four base sequences are determined.

For this purpose, four different vectors 1 to 4 are used to determine the respective DNA base sequences. In particular, the front single-stranded DNAa ₁ and the rear single-stranded DNAa _{2 of} the double-stranded DNAa and the front single-stranded DNAb ₁ and the rear single-stranded DNAb _{2 of} the double-stranded DNAb are combined in different vectors 1 to 4 in order to rearrange the vectors to build. These are four single-stranded DNA samples 5 through 8.

On the other hand, primers 9 to 12 are prepared which have different DNA base sequences, each of which can only hybridize with one type of vectors 1 to 4. The primers are each labeled with fluorophores with different emission wavelengths, including a fluorescein isothiocyanate with 515 nm emission wavelength (FITC, 4-fluoro-7-nitrobenzofurazan with 540 nm emission wavelength (NBD-F), tetramethylrhodamine isothiocyanate with 573 nm emission wavelength (TRITC) and Texas red with 605 nm emission wavelength. Then each of the DNA samples 5 to 8 is mixed together with the respective primers 9 to 12. Each primer 9 to 12 can only be hybridized with a corresponding one of the vectors 1 to 4. The obtained Hybridized DNA samples, as shown in Fig. 1, are DNA samples 5-9, 6- 10, 7-11 and 8-12 as a processed mixture.

Then the processed mixture is divided into four parts. Each part is composed individually with deoxynucleotide and dideoxynucleotide, so that the respective end group of the four parts can become adenine, cytosine, guanine or thymine. The results are subjected to respective enzymatic conversions (shown in FIG. 1 as A, C, G and T conversion) in order to carry out the complementary chain synthesis.

The complementary chain syntheses can produce individual DNA fragment groups whose end group of the DNA fragment is one of the four types. The DNA fragment groups include groups A, C, G and T. Group A (or A family) is the one in which the end group of the DNA fragment is adenine. In particular, the DNA fragment group A is a group in which the end groups of the DNA sample that has been hybridized with the vectors are adenine. It includes 5-9-A, 6-10-A, 7-11-A and 8-12-A, shown in Figure 1. The group C (or C family) is the one in which the end group of the DNA Fragments is cytosine. In particular, the group C DNA fragment is a group in which the end groups of the DNA samples hybridized with the vectors are cytosine. It includes 5-9-C, 6-10-C, 7-11-C and 8-12-C, shown in Fig. 2. The group G (or G family) is the one in which the end group of the DNA Fragments of guanine is. In particular, the DNA fragment group G is a group in which the end groups of the DNA samples hybridized with the vectors are guanine. It includes 5-9-G, 6-10-G, 7-11-G and 8-12-G shown in Fig. 2. The group T (or T family) is that in which the end group of the DNA Fragments is thymine. In particular, the DNA fragment group T is a group in which the end groups of the DNA samples hybridized with the vectors is thymine. It includes 5-9-T, 6-10-T, 7-11-T and 8-12-T, shown in Fig. 2.

In Fig. 3, in which the multi-color fluorescence detection type electrophoresis device is illustrated, an electrophoresis separating gel plate 110 contains 6% polyacrylamide, formed in a 0.3 mm space between 200 mm x 300 mm quartz plates (not shown). It should be noted that the concentration of the polyacrylamide is indicated herein as its total monomer concentration in% weight per volume (g / ml). The electrophoresis separation plate 110 has sample injection pockets 112, 113, 114 and 115 . The sample injection pockets each carry the DNA fragment groups A, C, G and T dripped there through an injection template or syringe in order to let them migrate. The electrophoresis separation gel plate 110 has a laser beam 123 directed through a side surface thereof to a portion about 25 cm below the bottom of the pockets to which the DNA fragment groups migrate using a laser beam source 116 . This causes the DNA fragment groups to emit fluorescent light. The emitted fluorescent light is divided by a prism (not shown) and spectra of different wavelengths corresponding to different fluorescent labels of samples 5 to 8 are obtained through optical bandpass filters. These fluorescence spectra are focused on a focusing part of a two-dimensional fluorescent light detector 117 in order to provide fluorescent light images corresponding to samples 5 to 8.

The fluorescent light images, which are focused on the focusing part of the two-dimensional fluorescent light detector 117 , can be displayed on a screen 121 as four line images 122 corresponding to the respective samples 5 to 8. At the same time, the fluorescent light images can be displayed by a display 120 , a controller 118 and a data processing unit 119 .

FIG. 4 shows an enlarged image of the line images 122 that are displayed on the screen 121 . The four line images correspond to the respective samples 5 to 8 downwards. Areas A, C, G and T indicated by dotted lines are areas corresponding to the respective DNA fragment groups A, C, G and T of each of the sample. The data obtained in this way can be processed arithmetically in order to determine the DNA base sequences simultaneously.

In this form of training, an argon laser with 488 nm wavelength and a He-Ne laser with 543 nm wavelength are used. The Argon laser and the He-Ne laser can be integrated in one unit to irradiate the same section or different sections, the 2 mm or more are spaced. For the suggestion of FITC and NBD-F the marking fluorophores the argon laser is used; for the suggestion of TRITC and Texas red, the He-Ne laser is used. For large samples additional fluorophores with longer wavelengths can be used will. Another He-Ne laser with 633 can be used for the additional fluorophores nm wavelength or a semiconductor laser can be used.  

The embodiment described above clearly shows that the The method according to the invention is advantageous in that so many samples can be edited like mixture forms themselves, the same time the operational effort can be reduced. In the process in which the DNA fragment groups A, C, G and T are marked with different colors and hike on the same path, the migration time differences have to be conditional be corrected by the color differences. The method according to the invention is Also advantageous in this regard because there is no correction of the migration speed differences required by the difference in marking dyes are conditional since the same sample is marked with the same color can be.

Claims (6)

1. A method for DNA base sequence determination, comprising the steps:

• i) cloning a large number of DNA samples, each with different vectors (1, 2, 3, 4);
• ii) Complementary chain synthesis of the samples (5, 6, 7, 8) obtained in step i) in a mixture with primers (9, 10, 11, 12), corresponding to the respective vectors (1, 2, 3, 4) , wherein the number of primers (9, 10, 11, 12) corresponds to the number of types of vectors (1, 2, 3, 4), each of these primers (9, 10, 11, 12) is able exclusively hybridize with a specific one of these vectors (1, 2, 3, 4), and the primers (9, 10, 11, 12) are labeled with fluorophores, each of which have different wavelengths;
• iii) Preparation of four DNA fragment groups, each with four different end groups, from the treated mixture (5-9, 6-10, 7-11, 8-12) obtained in step ii); and
• iv) electrophoretically migrating the four DNA fragment groups and detecting the fluorescent light emitted from the respective DNA fragments in each DNA fragment group.

2. A method for DNA base sequence determination, comprising the steps:

• i) cloning a multiplicity of different single-stranded DNA samples whose base sequence is to be determined, with respective vectors (1, 2, 3, 4), different for each sample;
• ii) labeling of the primers, the number of which corresponds to the number of types of vectors (1, 2, 3, 4), the base sequences of which are different from one another and which can only be hybridized with a specific vector, with respective fluorophores with different emission wavelengths;
• iii) hybridizing the primers (9, 10, 11, 12), labeled with the respective fluorophores with different emission wavelengths, with a multiplicity of corresponding vectors (1, 2, 3, 4) for the respective single-strand DNA samples (5, 6, 7, 8), cloned with the vectors (1, 2, 3, 4) in such a way that all the DNA samples (5, 6, 7, 8) cloned with the vectors (1, 2, 3, 4 ), mixed and processed with all the primers (9, 10, 11, 12);
• iv) divide each of the mixtures (5-9, 6-10, 7-11, 8-12) of the samples processed in step 3 into four parts;
• v) Complementary chain synthesis of the four-part mixtures (5-9, 6- 10, 7-11, 8-12) of the samples individually, so that end groups thereof can become adenine, cytosine, guanine or thymine, whereby individually a DNA Fragment group in which the end group of the DNA fragment is adenine, a DNA fragment group in which the end group of the DNA fragment is cytosine, a DNA fragment group in which the end group of the DNA fragment is guanine, and a DNA fragment group in which the end group of the DNA fragment is thymine; and
• vi) allowing the DNA fragment groups to migrate electrophoretically, detecting the individual fluorescent light emitted by the DNA fragment groups, and separating and detecting the samples according to their different emission wavelengths, namely from a large number of DNA samples.

3. A method for DNA base sequence determination according to claim 2, in which the Separation and the detection of the DNA fragment groups carried out in this way is that the samples are separated and detected using of the emitted wavelength differences in each DNA fragment group is moved electrophoretically on the corresponding migration paths and at the same time the variety of the end groups is specified by the Location of the migration path at which the fluorescent light is emitted, see above that a multitude of different base sequences of a single strand DNA sample is determined simultaneously.   4. A method for DNA base sequence determination according to claim 3, in which the samples are separated and detected by using fluorescent light wavelength differences of the respective DNA fragment groups that is emitted by applying a laser beam ( 123 ) to sections with certain distances from which the Hike DNA fragment groups on the migration tracks. 5. A method for DNA base sequence determination according to claim 4, in which the separation and the detection of the samples is carried out using the fluorescence light emission wavelength differences of the DNA fragment groups in such a way that the fluorescence light of the DNA fragment groups is separated with a wavelength separator and each of the separated wavelengths is individually detected by a detector ( 117 ). 6. The method for DNA base sequence determination according to claim 5, in which the detector ( 117 ) is a two-dimensional fluorescent light detector.