DE3930312A1 - METHOD FOR DIRECTLY SEQUENCING NUCLEIC ACID CHAINS - Google Patents

Abstract

A method for direct sequencing of nucleic acid chains by denaturation followed by amplification of the regions to be sequenced by repetition of a cycle includes a) a nucleic acid synthesis step in which the nucleic acid to be sequenced is replicated, as a matrix strand in the presence of the four deoxynucleoside triphosphates, by a polymerase, starting from oligonucleotide primers which are complementary to the 3' ends of the antiparallel strands of the nucleic acids to be sequenced; followed by b) a denaturation step. The ends of the resulting nucleic acids are marked during or after amplification. Sequencing of the marked nucleic acids is carried out by incorporating modified nucleotides at positions speicific for a particular base, shortening the amplified nucleic acids to these respective positions and separating the resultant nucleic acid fragments by electrophoresis. The modified nucleotides are incorporated during amplification in four different cycles by using a deoxynucleoside- alpha -thiotriphosphate together with each of the three other unmodified deoxynucleoside triphosphates instead of the four deoxynucleoside triphosphates.

Description

The invention relates to a method for direct Sequencing of nucleic acid chains by denatury tion and then amplification of the Be to be sequenced range by going through a cycle several times, full

• a) a nucleic acid synthesis step in which the sequencing nucleic acid as a template strand in Presence of the four deoxynucleoside triphosphates from a polymerase starting from oligonucleo tid primers to the 3'-ends of the antiparallel Strands of the nucleic acids to be sequenced are complementary, is replicated, followed by
• b) a denaturation step,

the nucleic acids formed during or after the amplification is marked at one end, Sequencing of the labeled nucleic acids by incorporation modified nucleotides each for one base specific positions, shortening of the amplified Nucleic acids each up to these positions and electrophoretic separation of the resulting nucleus acid fragments.

Analysis of the molecular basis of genetic Diseases, the study of polymorphisms, the Examination of DNA for phenotypic mutations just a few of the tasks for which an exact finding development of the nucleotide sequence of nucleic acids is essential is. However, such investigations often stand only small amounts of nucleic acid available, what is not sufficient for sequencing according to the  common techniques. Another problem is the Presence of additional foreign nucleic acid material, which is the sequencing of a particular nucleic acid sequence difficult. A big step forward in this area is that of Saiki et al., Science 230 (1985), 1350-1354 developed so-called polymerase chain reaction (PCR) - Technology based on small amounts of nucleic acid an exponential specific synthesis of the desired Nucleic acids or segments thereof allowed. At this Technique based on two primers are preferred short synthetic oligonucleotide chain extension reactions carried out, the two primers to the 3'-ends of the antiparallel strands of the desired DNA segments are complementary. These chain extensions reactions are carried out several times, whereby during the extension cycles are denatured by heating, whereby the chain extension reaction formed nucleic acids from the original one Solve a template DNA and this for a new synthesis make round accessible. That way you can large amounts of the nucleic acid or nucleotide sought acid fragments synthesized in a relatively short time will.

Known methods for sequencing nucleic acids are for example the sequencing of Maxam and Gilbert, Methods Enzymol. 65: 499-560 (1980) Sanger et al., Proc. Natl. Acad. Sci. UNITED STATES. 74 (1977), 5463 or the method of G. Gish and F. Eckstein, Science, Vol. 240 (1988) 1520-1522. With the last method is called from a primer chain synthesis similar to the Sanger-Me method, but instead of four different ones  Dideoxynucleotides are the corresponding deoxynucleoside thio triphosphate used, and then a partial strand break at the thiophosphate positions of the formed Chemically effects nucleic acids.

Sequencing of DNA amplified by PCR is, for example, according to the Sanger method of Saiki et al., Science, Vol. 239 (1988), pages 487 to 491. However, this method has the disadvantage that from the amplified DNA using a additional primers specifically for the four Nucleotide DNA copies using the chain termination method have to be manufactured.

Sequencing of PCR-amplified DNA after the Maxam-Gilbert method is from Keohavong et al., In DNA, Vol. 7, No. 1, (1988), pages 63 to 70. However, this method also has the disadvantage that that after amplification a normal one sequencing using specific Mo dification reactions for the four different ones NEN nucleotides must be carried out.

The invention was therefore based on the object Provide method that only allows ge small amount of nucleic acids present particularly quickly and easy to sequence.

According to the invention, this object is achieved by a Method for direct sequencing of nucleic acid chains by denaturing and then amplifying the Areas to be sequenced by repeatedly run a cycle, comprehensively  

• a) a nucleic acid synthesis step in which the sequencing nucleic acid as a template strand in Presence of the four deoxynucleoside triphosphates from a polymerase starting from oligonucleo tid primers to the 3'-ends of the antiparallel Strands of the nucleic acids to be sequenced are complementary, is replicated, followed by
• b) a denaturation step,

wherein the resulting nucleic acids are labeled at one end during or after the amplification, sequencing of the labeled nucleic acids by incorporating modified nucleotides at positions specific to a base, shortening of the amplified nucleic acids to these positions and electrophoretic separation of the resulting nucleic acid fragments, which characterized in that one uses a deoxynucleoside α- thio-triphosphate together with the three other unmodified deoxynucleoside triphosphates instead of the four deoxynucleoside triphosphates for modification in the amplification in four different batches.

The method according to the invention enables rapid and accurate sequencing in just one step, with the cumbersome sequencing methods described in Ver binding with polymerase chain reactions so far in the state the technology are described can be avoided.

In one embodiment of the method according to the invention rens are in the amplifi after chain reaction ornamentation cycles in the four polymerization batches amplified nucleic acids precipitated and if necessary triphosphates not incorporated according to known per se  Methods, e.g. B. chromatographically separated. On in conclusion, nucleic acid fragments with less Length in the usual way, e.g. B. gel electrophoretic, be separated. By adding an alkylating agent the largest obtained in the separation Fragment followed by heating becomes a partial Cleavage of the nucleic acids at the thiophosphate position NEN of the nucleic acids. Under elevated temperature is a temperature in the context of the invention understood that lead to a partial strand break at the modified places leads. Preferably in Heated to between 80 ° C and 95 ° C within the scope of the invention. Due to the split conditions (see also Gish and Eckstein supra) is amplified in the Statistically nucleic acids at each thiophosphate posi tion split at least once, the four approaches, each containing different labeled nucleic acid fragments, placed side by side on a sequencing gel gene, the nucleic acid fragments, depending on the type the marking, according to methods known per se (e.g. Autoradiography) and the nucleus acid sequence read.

As alkylating agents, those from the prior art Known alkylating agents can be used. Preferred alkylating agents according to the invention here 2-iodoethanol, 2,3-epoxy-1-propanol, 2,3-epoxy propyltrimethylammonium chloride, iodoacetic acid, iodoacet amide or cyanogen bromide, of which again in particular preferably use 2-iodoethanol and 2,3-epoxy-1-propanol be det.

Other alkylating agents preferred according to the invention are methyl iodide, dimethyl sulfate and ethyl iodide. At However, the use of these must precede the pH the temperature increase to about 90 ° C by adding a strong base, preferably to a value of at least 11.  

Alternatively, iodine / ammonia or H ₂ O ₂ can also be used as cleavage reagents instead of an alkylating agent.

In a further embodiment of the invention The procedure is after amplification by the Polyme rase chain reaction no time-consuming cleaning of the amplified nucleic acids by separation of not built-in nucleotides and / or fragments with low length required.

Surprisingly, it was found that at amplification alongside the expected product full length a variety of shorter nucleic acids is synthesized with different lengths. These shorter fragments can already be relative small number of amplification cycles observed will. The emergence of such a fragment mixture is amplified under different Reaction temperatures (e.g. 59 ° C or 71 ° C) or below different duration of the amplification cycles (e.g. 3 minutes or 7 minutes), as well as in and in Absence of thiophosphates found. Because of this surprising observation it is possible to make a legible sequence ladder by direct removal of the ends of the nucleic acid mixture in the 3 ′ → 5 ′ direction in each case to get up to a thiophosphate.

To carry out such a reaction, 3 ′ → 5′- Suitable for exonucleases, the thiophospho diester compounds only very bad compared to normal phosphodiester Can split bonds. Sequencing method based on based on this principle are already known (Labeit et al., (1986) DNA 5, 173-177; PCT application PCT / GB86 / 00349 from the company AMERSHAM). Happens according to these procedures  the polymerization reaction by the Klenow fragment the E. coli DNA polymerase I, being in the DNA chains small amounts of thiophosphate can be incorporated. The Degradation in the 3 ′ → 5 ′ direction occurs through exonuclease III. In this case, substrates are DNA chains full length and a statistical installation by Thiophos phate. In contrast, in the method according to the invention Mixture of DNA fragments of different lengths and a complete incorporation of thiophosphate Substrate for the exonuclease.

However, the exo is for the method according to the invention nuclease III not very suitable as some bands on it the sequence leader has a low intensity and therefore no legible base sequence can be obtained can. Very suitable for the method according to the invention surprisingly, the snake venom Phosphodiesterase, a 3 ′ → 5′-exonuclease, from the is known to remove thiophospho diester compounds cleaves the factor 100 more slowly than the phosphodiester bin dungen (Burgers and Eckstein (1979), Biochemistry 18, 592-596). Particularly suitable for the invention Procedure turns out when the breakdown of the nucleus acid end by snake venom phosphodiesterase a reaction temperature of 16 ° C takes place. If on 5'-end there is a marker group that one Prevents degradation from the 5'-end, can also 3 ′ → 5′-exonuclease used with a 5 ′ → 3 ′ exonuclease activity is associated.

The method according to the invention thus discloses one Sequencing method as quickly as possible to know the sequence of a particular nucleic acid leads and is also particularly easy to carry out.  

It was highly surprising that the deoxynucleoside α- thiotriphosphates already used in the sequencing method of Gish and Eckstein as substrates in the polymerase chain reaction did not cause any deterioration in the yield or the accuracy of the replication and therefore two reactions, namely the amplification and sequencing can be carried out simultaneously.

The polymerization chain reaction is as above already executed, denatured after each cycle to the newly formed nucleic acid strands from the template strain to separate. This will make the old matrix stem for a new round of synthesis accessible, as well as the new ones already formed in a previous cycle Nucleic acids. This is done by warming up to more than 90 ° C, but this makes non-heat-stable polymerases be deactivated. Therefore, when using not heat stable polymerases new for each cycle Polymerase can be added. It is therefore fictional preferred according to the polymerization chain reaction to use a heat-stable polymerase for DNA syn preferably the Taq polymerase to thereby achieve the To further facilitate procedures.

In the polymerase chain reaction according to the invention preferred for denaturation in each cycle to 95 to Heated to 96 ° C. Here, a particularly high degree of Denaturation reached, so that the chain reaction be can be carried out particularly efficiently; on the on the other hand, the heat-stable preferred Taq poly merase is not inactivated at these temperatures.

According to the invention, it is preferred to have 12 to 30 polymers sation cycles, this is usually off sufficient nucleic acid.  

It is also preferred according to the invention to use primers Use 10 to 25 nucleotides in length.

It is suitable for the course of the polymerization cycles for example when using two 18-mer oligonucleos tide after the addition of polymerase and nucleotides, the sequence: 3 minutes 71 ° C, 1 minute 95 to 97 ° C and 2 minutes 37 ° C. The last cycle is the period prolonged to 7 minutes at 71 ° C and then to room temperature let the temperature cool. It is preferred that the ange given steps in a well insulated temperature control system so that no uncontrolled Ab deviations from the desired temperatures occur.

Preferred especially for longer amplification In contrast, nucleic acids (greater than about 300 bp) is a Polymerization cycle with the sequence: 7 minutes 55 ° C, 1 minute 95 to 97 ° C and 2 minutes 37 ° C.

The labeling of the nucleic acids for later visibility Preparation of the obtained, separated by electrophoresis According to the invention, nucleic acid fragments can be the same take place in time with the amplification reaction, or but only after the amplification reaction. Here is in principle it is possible to use both strands of nucleic acids to mark, but this will make it necessary, after the Amplification reaction and before treatment with Alkylating agents and partial cleavage the two complementary strands, for example by gel electro separate phorese, and the respective single strands separately or just one of the two complemen tary strands are then treated with the alkylating agent deln. Since this is relatively expensive, it is fiction according to preferred, only one of the two complementary To mark nucleic acid strands. There is again around the possibility during or after the amplification  reaction to carry out the marking. Usable are known in the sense of the invention Methods for labeling nucleic acids.

According to the invention, radioactive labels are preferred, Label with a fluorescent dye or Enzyme that is a chromogenic substrate under development a recognizable color reaction splits. In the sense of the Invention is under a label with an enzyme also understand a label about a conjugate, where the one partner of the nucleic acid Binding system is coupled, and the enzyme with the other partner of the binding system. As The binding system comes with known binding systems such as biotin / (strept) avidin or hapten / anti gene binding pairs in question.

For marking during amplification, inventions are made According to the use of one of the primers in marked form det. A radioactive is particularly preferred for this labeled primer used.

For marking after the amplification is fiction according to preferably a primer in at its 5'-end phosphorylated form used for amplification and then the other, non-phosphorylated primer with Radioactive phosphorylated with the help of a kinase. There are however, other methods of markie known per se tion of nucleic acids applicable for sequencing.

The following examples in conjunction with Figs. 1 to 3 further illustrate the invention.

Fig. 1 shows the autoradiograph of a sequencing gel of sequencing a nucleic acid according In Game 1.

Fig. 2 shows the gel electrophoretic separation of amplified nucleic acids as a function of the number of reaction cycles.

Fig. 3 shows the autoradiograph of a sequencing gel of sequencing a nucleic acid according to Example 5.

example 1

Sequencing of labeled after the chain reaction DNA by partial cleavage after alkylation with 2,3-ethoxy-1-propanol.

• a) Phosphorylation of the oligonucleotide primer
  An oligonucleotide primer with the sequence 5 '- [AGGGTTTTCCCAGTCACG] was phosphorylated before PCR amplification, as described by Taylor et al., Nucleic Acids Res. 13, (1985) 8765-8785. For this purpose, 40 ug oligonucleotide with 30 U (units) of polynucleotide kinase in a reaction mixture (30 ul), composed of 100 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol, 10 mM MgCl ₂ and 1 mM ATP were treated. After two hours at 37 ° C, the reaction mixture was heated to 70 ° C for 15 minutes and the product purified using a SEP-PAK-C18 cartridge (Waters Associates) as described by Ott and Eckstein, Biochemistry 26 (1987), 8237 -8241, described, cleaned. The phosphorylated oligonucleotide was stored at -20 ° C as an aqueous solution with a concentration of 10 A ₂₆₀ units per ml.
• b) polymerase chain reaction
  The polymerase chain reaction amplification was carried out manually. BglI-digested linear double-stranded M13 mp18 DNA was in an amount of 200 ng in 320 ul of a solution containing 63 mM KCl, 12.5 mM Tris-HCl, pH 8.4, 0.016% (w / v) glycerol, 3.8 mM MgCl ₂ , 0.8 µM (0.07 A ₂₆₀ units) 5′-phosphorylated oligonucleotide primer and 0.8 µM (0.07 A ₂₆₀ units) of the non-phosphorylated oligonucleotide primer 5′-d [CACCCTGGCGCCCAATAC] - 3'resolved. Hybridization of the oligonucleotides was carried out by heating at 96 to 97 ° C for 5 minutes to denature the DNA and then cooling to 37 ° C for 5 minutes. The solution was divided (80 μl) into four 750 μl Eppendorf vessels, diluted to 100 μl with a mixture of 3 deoxynucleoside triphosphates and an S _p -De oxynucleoside 5′-O- (1-thiotriphosphate), which according to Taylor et al., Nucleic Acids Res. 13 (1985) 8749-8764, to give a final concentration of 250 µM of each of the deoxynucleotides and four units of Taq DNA polymerase were added.
• 100 μl of paraffin were added to the reaction mixture oil layered. The reactions became a temp maturation cycle of 71 ° C for 3 minutes, 96 to 97 ° C  15 cycles for one minute and 37 ° C for two minutes len subjected. For the last cycle, the Reaction time at 71 ° C extended to 5 minutes, then the samples were allowed to cool to room temperature calmly. The paraffin oil was pipetted off and by washing twice with ethyl ether (each 200 µl) completely removed. The amplified Product could easily be in a 1.5% agarose gel made visible by ethidium bromide, about 8 µl of the mixture is applied to the gel were.
• c) Labeling of the amplified fragments Each amplified product was purified on a Sepha dex G50 centrifuge column (approx. 1 ml) equilibrated with H ₂ O in order to remove excess nucleotides and salt. The flow through the column was precipitated by adding 10 μl of 3M sodium acetate, pH 6.0 and 500 μl of absolute ethanol. After cooling to -78 ° C for 15 minutes, followed by centrifugation for 15 minutes, the supernatant was decanted off and the pellet was washed with 57 µl of 70% ethanol and then dried.
• 30 μCi [ γ32 P] ATP were added to each pellet and the solutions were dried again. Each pellet was taken up in 4.5 ul 20 mM Tris-HCl, pH 8.0, 20 mM MgCl ₂ and 2.4 mM 2-mercaptoethanol. For this purpose, 15 U (units, 0.5 μl) of T4 polynucleotide kinase were added and the reaction was carried out for 45 minutes at room temperature. The reaction mixture was mixed with 2 μl of a stop mixture (96% formamide, 10 mM EDTA, 0.1% (w / v) bromophenol blue and 0.1% (w / v) xylenecanol ff) and poured onto an 8% polyacrylamide Sequencing gel applied.
• After electrophoresis of the amplified fragments was obtained by exposure to a Kodak X-Omat  XAR-5 Films visualized the DNA. The question elements were presumably due to two gangs seen from incomplete denaturation. Both Bands were cut out and the DNA from the Polyacrylamide gel using the method of G.M. Rubin, Methods in Cell Biology, Vol. XII, Yeast Cells (1975) Ed. by. DM. Prescott, pages 45-85, Academic Press, N.Y. extracted.
• d) sequencing reaction
  The radiolabelled fragments were dissolved in sufficient water to give approximately 500 cps (per µl of solution) (as determined by measurement with a Geierger counter). For each sequencing reaction, 4 ul DNA was mixed well with 2 ul stop mixture (see above) containing 0.5% (v / v) 2,3-ethoxy-1-propanol. This stop mix solution was freshly prepared before the experiment to rule out any possibility of hydrolysis of the alkylating reagent. The samples were heated to 95 ° C for 3 minutes and then cooled on ice before being applied to an 8% polyacrylamide sequencing gel. After electrophoresis, the gel was dried and a Kodak-X-Omat-XAR-5 film was exposed for 48 hours without an intensifying screen. Fig. 1 shows the autoradiography of this sequence gel.

Example 2

Sequencing of labeled during the chain reaction DNA by partial cleavage after alkylation with 2,3-ethoxy-1-propanol

• a) Radioactive labeling of the oligonucleotide primer
  The oligonucleotide primer (4 μg) used in Example 1a) was phosphorylated in a 30 μl reaction solution which was composed of 100 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol, 10 mM MgCl ₂ , 60 μ Ci [ γ - ³² P] ATP and 60 units of T 4 polynucleotide kinase. After 45 minutes at 37 ° C, the reaction mixture was heated to 70 ° C for 15 minutes and the product was purified using a SEP-PAK C 18 cartridge (Waters Associates). The radioactively labeled oligonucleotide was stored as an aqueous stock solution with 3.6 A ₂₆₀ units per ml and had a specific activity of 3 × 10 ⁹ cpm per A ₂₆₀ units.
• b) polymerase chain reaction and sequencing
  The polymerase chain reaction was carried out as in example 1b), with the exception that the radio-labeled primer was used instead of the phosphorylated unlabeled primer. After 15 cycles, the paraffin oil was removed and the DNA was precipitated by adding 10 µl of 3 M sodium acetate, pH 6.0 and 250 µl of absolute ethanol by cooling to -80 ° C for 30 minutes followed by 20 minutes of centrifugation. Excess ethanol was removed by drying the pellets for two minutes and the DNA was dissolved in 20 μl mM Tris-HCl, 0.1 mM EDTA, pH 8.0 and 2 μl stop mixture (see above). The amplified fragments were purified on an 8% polyacrylamide sequencing gel as described above. The isolation of the DNA from the gel and the sequencing reaction were carried out as described in Example 1.

Example 3

Sequencing by partial cleavage after alkylation with dimethyl sulfate
Radioactive labeled polymerase chain reaction fragments prepared as in Example 1a) through c) were dissolved in sufficient water to give approximately 500 cps / µl solution (as determined by measurement with a Geiger counter). For each sequencing reaction, 4 ul of the phosphorothioate-containing DNA was diluted with 2 ul 0.5% (v / v) aqueous dimethyl sulfate freshly prepared before each experiment and allowed to stand for 10 minutes at room temperature. For the DNA cleavage, 2 μl of 0.5 N sodium hydroxide were added and the samples were heated at 95 ° C. for three minutes for three minutes. Before the electrophoresis, 2 μl of the stop mixture (96% formamide, 10 mM EDTA, 0.1% (w / v) bromophenol blue and 0.1% (w / v) xylene cyanol ff) were added, the samples again for three minutes at 95 Heated to ° C, cooled on ice and subjected to gel electrophoresis as described under 1d).

Example 4

Analysis of amplified nucleic acids depending on the number of reaction cycles
M 13mp18 single-stranded DNA was amplified over 21 cycles of the polymerase chain reaction with Taq DNA polymerase from Cetus in the presence of 250 μmol / l each of dCTP α S, dATP, dGTP and dTTP. As a non-radioactive labeled oligonucleotide primer

5 ′ - [CAC CCT GGC GCC CAA TAC] -3 ′,

marked as ³²P Oligonucleotide primer

5 ′ - [TTA CGC CAG CTG GCG AAA] -3 ′

used. In 50 ul reaction volume were 50 ng DNA, 0.025% Nonidet P-40 (Sigma), 0.025% Tween 20 (BioRad), 2.5 mmol / l MgCl ₂ , 10 mmol / l Tris-HCl, pH 8.4 and 2 pmol of each primer (0.1 A ₂₆₀ units per ml) included. The reaction solution was overlaid with 100 ul paraffin oil. The reaction was carried out in a Techne PHC-1 programmable driblock under the following conditions: 55 ° C, 7 minutes; 96 ° C, 1.5 minutes; 37 ° C, 2.5 minutes over 21 cycles, followed by heating to 55 ° C for 9.9 minutes. After the reaction has ended, the paraffin oil is removed and the samples are extracted with 400 μl water saturated ether.

5 μl aliquots were removed from the reaction mixture immediately after addition of the enzyme ( FIG. 2, lane a ) and after three amplification cycles (lanes b to h ).

Fig. 2 shows the product distribution as a function of the number of reaction cycles. Nucleic acids that were smaller than the expected product (indicated by an arrow) appeared after only a few cycles.

After 21 cycles, the expected product was 384 bp While length was the main product of the reaction, it was however, termination products of almost any length are available the. A corresponding experiment with the Taq polymerase from Amersham brought the same result.

Example 5

Sequencing of labeled during the chain reaction DNA by degrading the nucleic acid ends with snake venom Phosphodiesterase

The same DNA as in Example 4 was used for sequencing used. The amplification reaction was in the presence marked by ³²P

5 ′ - [AGGGTTTCCCAGTCACG] -3 ′,

250 µmol / l of 3 deoxynucleoside triphosphates, each 250 µmol / l of an α -thiodeoxynucleotide ( Fig. 3, lanes A , C , G and T ) and otherwise carried out under the same conditions as described in Example 4.

After removing the paraffin oil, however, was immediately too 5 µl each of a reaction mixture 1 µl of a 1: 10- Dilution of a 2 ng / ml snake venom phosphodieste rase solution (Boehringer Mannheim). Samples were then incubated at 16 ° C for 15 minutes. The Reactions were stopped by adding a stop buffer (96% Formamide, 10 mmol / l EDTA, 0.1% bromophenol blue and 0.1% (w / v Xylencianol FS) and heating for 3 minutes ended at 95 ° C. The samples were separated on an 8% polyacrylamide gel.

Fig. 3 shows a legible sequence ladder, which was obtained by treatment of nucleic acid fragments from an amplification reaction with snake venom phosphodiesterase.

Claims (22)

1. A method for direct sequencing of nucleic acid chains by denaturing and then amplifying the areas to be sequenced by repeating a cycle several times, comprising

• a) a nucleic acid synthesis step in which the nucleic acid to be sequenced stranded as templates in the presence of the four deoxynucleoside triphosphates from a polymerase, starting from oligonucleotide primers which are complementary to the 3′-ends of the antiparallel strands of the nucleic acids to be sequenced, is replicated, followed by
• b) a denaturation step,

wherein the nucleic acids formed are labeled during or after amplification, at its one end, sequence of labeled nucleic acids by incorporation of modified nucleotides at each specific for a base positions, shortening of the amplified nucleic acids up to these positions and electrophoretic Auftren voltage of the nucleic acid fragments formed, characterized characterized in that one uses instead of the four deoxynucleoside triphosphates one deoxynucleoside α- thio-triphosphate together with the three other unmodified deoxynucleoside triphosphates for the incorporation of modified nucleotides in the amplification in four different batches. 2. The method according to claim 1, characterized characterized that one after an alkylating agent in the amplification step admits and partial split at the positions of the modified nucleotides. 3. The method according to claim 2, characterized characterized that one after nuc not built into the amplification step leotides and nucleic acid fragments with less Length separates. 4. The method according to claim 2 or 3, characterized characterized that one as Alkylating agent 2-iodoethanol, 2,3-epoxy-1-propa nol, 2,3-epoxypropyltrimethylammonium chloride, Iodoacetic acid, iodoacetamide or cyanogen bromide used. 5. The method according to claim 4, characterized characterized that one as Alkylating agent 2-iodoethanol or 2,3-epoxy-1-pro panol used. 6. The method according to claim 2 or 3, characterized characterized that one as Alkylating agent dimethyl sulfate, methyl iodide or ethyl iodide and in addition the pH in the batch before heating by adding a strong base increases. 7. The method according to claim 6, characterized characterized in that the pH increased to at least 11.   8. The method according to claim 1, characterized characterized that one after the amplification step directly a 3 ′ → 5'-exonuclease admits and degradation to the Positions of the modified nucleotides causes. 9. The method according to claim 8, characterized characterized that one as Use snake venom phosphodiesterase exonuclease det. 10. The method according to claim 9, characterized characterized that one the Incubation with snake venom phosphodiesterase at a temperature around 16 ° C. 11. The method according to any one of claims 1 to 10, characterized, that a heat stable polymerase is used. 12. The method according to claim 11, characterized characterized that one the Taq polymerase used. 13. The method according to claim 12, characterized characterized that one the Amplification in cycles of 7 minutes each 55 ° C. 14. The method according to any one of claims 1 to 13, characterized, that for denaturation in each cycle to 95 heated to 96 ° C.   15. The method according to any one of claims 1 to 14, characterized, that you do 12 to 30 cycles. 16. The method according to any one of claims 1 to 15, characterized, that primers with a length of 10 to 25 Nucleotides used. 17. The method according to any one of claims 1 to 16, characterized, that the radioactive amplified nucleic acids tiv, with a fluorescent dye or Enzyme labeled. 18. The method according to claim 17, characterized characterized that one only marked one of the two nucleic acid strands. 19. The method according to claim 18, characterized characterized that one for Label one of the during amplification Primer used in marked form. 20. The method according to claim 19, characterized characterized that one radioactively labeled primer used. 21. The method according to claim 20, characterized characterized that one for Label after amplification one of the at the primer in at its 5'-end phosphorylated Form used for amplification and then the other, non-phosphorylated primers with the help a radioactive phosphorylated kinase.