SE456348B - PROCEDURE FOR ELECTROPHORETIC ANALYSIS OF DNA FRAGMENTS - Google Patents

Description

456 348 to allow chemical analysis. Another method was the near perfection of gel electrophoretic methods for high-resolution separation of oligonucleotides based on their size.

However, the conceptual key development was the introduction of methods to generate size-nested sets of fragments of cloned, purified DNA which in their length collection contain the information required to define the nucleotide sequence comprising the parent DNA molecules. Two different methods for generating these fragment sets were widely used, one of which was developed by Sanger; Sanger, F., Nicklen, S. and Coulson, A.

R., Proc. Natl. Acad. Sci. USA 74, 5463 (1977) and Smith, A.

J. H., Methods in Enzymology 65, 56-580 (1980): and the other by Maxam and Gilbert; Maxam, A. M. and Gilbert, W., Methods in Enzymology 65, Q99-559 (1980).

The method developed by Sanger is called the dideoxy chain termination method. In the most widely used variation of this method, a DNA segment is cloned into a single-stranded DNA phage, such as M13. These phage DNAs can serve as templates for the primed synthesis of the complementary strand with the Klenow fragment of DNA polymerase I. The primer is either a synthetic oligonucleotide or a restriction fragment isolated from parent recombinant DNA, which specifically hybridizes to a region of the M13 vector near the 3 'end of the cloned inset. In each of the four sequencing reactions, the primed synthesis is performed in the presence of a sufficient amount of the dideoxy analog of one of the four possible deoxynucleotides, so that the growing chains are randomly terminated by the incorporation of these "dead-end" nucleotides. of dideoxy to deoxy forms are adjusted to provide a spread of termination outcome corresponding to all conceivable chain lengths that can be resolved by gel electrophoresis.The products from each of the four primed synthesis reactions are then separated on individual bands of polyacrylamide gels by electrophoresis.

Radioactive markers incorporated into the growing chains were used to generate an autoradiogram image of the pattern of DNA in each electrophoresis band. The sequence of the deoxynucleotides in the cloned DNA is determined by examining the pattern of the bands in the four folds.

The method developed by Maxam and Gilbert uses chemical treatment of purified DNA to generate sized sets of DNA fragments analogous to those produced according to the Sanger method.

Single or double stranded DNA, labeled with radioactive phosphate at either the 3 'or 5' end, can be sequenced according to this method. In four reaction cycles, cleavage at one or two of the four nucleotide bases is induced by chemical treatment. The cleavage consists of a three-step process: modification of the base, removal of the modified base from its sugar and strand splitting at this sugar. The reaction conditions are adjusted so that the majority of the end-labeled fragments generated are of the order of magnitude (generally 1 to 400 nucleotides) that can be resolved by gel electrophoresis. Electrophoresis, autoradiography and pattern analysis are performed mainly according to Sanger methods. (Although chemical fragmentation each time necessarily yields two bits of DNA, only the bit containing the end marker is detected on the autoradiogram.) Both of these DNA sequencing methods are widely used and each has many variations. In each method, the sequence length that can be obtained from a single round of reactions is limited primarily by the solubility of the polyacrylamide gels used for the electrophoresis. In general, 200 to 400 bases can be read from a simple set of gel tapes. Although successful, both methods have serious drawbacks, problems primarily associated with the electrophoretic process. One problem is that radiolabeling must be used as a marker for the localization of the DNA bands in the gels, which causes difficulties due to the short half-life of phosphorus-32 and the consequent instability of the radiolabeled reagents as well as problems with ring and management of radioactive waste. Of greater importance 456 348 is that the nature of the autoradiography (the film image of a radioactive gel band is wider than the band itself) and the comparison of the band positions between four different gel bands (which may appear uniform or non-uniform from a band mobility point of view) may limit the observed resolution of the bands. and thus the sequence length that can be read from the gel. Furthermore, track-to-track irregularities make automatic scanning of autoradiograms difficult - the human eye can currently compensate for these irregularities much better than computers.

This need for manual "reading" of the autoradiograms is time consuming, tedious and easily gives incorrect results. In addition, one cannot read the gel patterns during the electrophoresis to be able to terminate the electrophoresis as soon as the solution becomes insufficient to separate adjacent bands, but one must terminate the electrophoresis at some standard time and wait for the autoradiogram to be developed before the sequence reading can begin. .

The present invention overcomes these and other problems associated with the electrophoresis step of the DNA sequencing procedures and represents a significant advance in the art.

In summary, the present invention encompasses a novel method of electrophoretic analysis of DNA fragments prepared in DNA sequencing methods, wherein at least one chromophore or fluorophore is used to label the DNA fragments generated by the sequencing chemistry and enables the detection and characterization of the fragments as they are cleaved. by electrophoresis through a gel. The detection uses an absorption or fluorescence photometer capable of monitoring the labeled bands as they move through the gel.

The present invention also encompasses electrophoretic analysis of DNA fragments prepared by DNA sequencing methods comprising: - establishing a source of chromophore- or fluorescently labeled DNA fragments from the sequencing methods, - establishing a zone for containing a electrophoresis gel, - establishes devices for introducing said labeled DNA fragments into said zone, - establishes a photometric device for monitoring "said labeled DNA fragments as they move through said gel, and - monitors said labeled DNA fragments as they are introduced in said zone and moves through said gel.

An object of the present invention is to provide a new method for sequence analysis of DNA.

Another object of the present invention is to provide a novel system for analyzing DNA fragments.

A particular object of the present invention is to provide an improved method for sequencing DNA.

These and other objects and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings.

Referring to the drawings: Figure 1 is an illustration of a device for end-labeling a DNA fragment with a fluorescent marker. Pst. I and T4 DNA ligases are enzymes commonly used in recombinant DNA research.

Figure 2 is a block diagram of an automated DNA sequencer, gel electrophoresis system.

Figure 3 is a schematic diagram of an optical configuration in the detector unit. P, lamp source; Ll, objective lens; L2, collision 456 348 mation lens; F1, UV blocking filter; P2, heat-blocking filter; F3, "band pass excitation" filter; F4, "long pass emission" filter: DM, dichroic mirror; C, polyacrylamide gel; PMT, photomultiplier tube.

Figure 4 is a schematic diagram of another optical configuration in the detector unit. F1 to F4 are "bandpass" filters centered at the emission maximum for the different colors. P1 to P4 are photomultiplier tubes. The excitation light is of such a wavelength that it is not transmitted through any of the filters F1 to F4.

Figure 5 is a comparison of the data types obtained by DNA sequencing of the sequence shown in Figure 1.

Figure 6 is a block diagram of a preferred DNA sequencer of the present invention.

Design of labeled primers for use in the Sanger method.

In the previous methods of DNA sequencing, including those based on the Sanger dideoxy chain termination method, simple radiolabeled phosphorus-32 was used to identify all bands on the gels. This makes it necessary for the fragment sets generated in the four synthesis reactions to be run on separate gel bands and poses the problems associated with comparable band mobilities in the different bands. These problems are overcome according to the present invention by using a set of four chromophores or fluorophores with different absorption and fluorescence maxima, respectively. Each of these markers is chemically linked to the primer used to initiate the synthesis of the fragment strands. Each labeled primer is then in turn paired with one of the dideoxynucleotides and used in the primed synthesis reaction with the K1enow fragment of DNA polymerase. 456 348 The primers must have the following characteristics. l) They must have a free 3'-hydroxyl group to allow chain extension through the polymerase. 2) They must be complementary to a unique 3 'region of the cloned inlet. 3) They must be long enough to hybridize to form a unique, stable duplex. 4) The chromophore or fluorophore must not interfere with the hydridization or prevent 3 'end extension by the polymerase.

The above conditions 1, 2 and 3 are met by several synthetic oligonucleotide primers, which are commonly used for Sanger-type sequencing using N13 vectors. One such primer is 12 more 5'TCA CGA CGT TGT 3 ', where A, C, G and T represent the four different nucleoside components of DNA; A, adenosine; C, cytosine; G, guanosine; T, Lymidine.

The chemistry for the coupling of the chromophoric or fluorophoric markers is described in the applicant's Swedish patent application No. 8406484-9 (filed December 19, 1984), the contents of which are expressly incorporated herein. The strategy used is to introduce an aliphatic amino group at the 5 'end as a final addition in the synthesis of the oligonucleotide primer. This reactive amino group can then be easily coupled with a variety of amino-reactive chromophores and / or fluorophores. This approach contributes to the compatibility of the labeled primers with condition 4 above.

The four dyes used must have high extinction coefficients and / or reasonably high quantitative yields for fluorescence. They must have well-resolved absorption maxima and / or emission maxima. A representative set of four such amino-reactive dyes are: fluorescein isothiocyanate (FITC, Äššx = 495, Äšïx = 520, E495 f 8 x l04), eosin isothiocyanate (EITC, Äššx = 522, Äšrâx = 533, 6522 e * 104), tetramethylrodamine isothiocyanate (TMRITC, 'Ää: x = 550, Äââx = 578, O 5 4 x 104), and substituted rhodamine isothiocyanate (XRITC, X _ 580, Äšïx = 604, 5580 =' sx 104), where k represents the wavelength in nanometers, Ex is excitation, Em is emission, max s 456 348 is maximum and 5 is the molar extinction coefficient.

These dyes are attached to the M13 primer and the conjugates are electrophoresed on a 20% polyacrylamide gel. The labeled primers are visible both through their absorption and fluorescence in the gel.

All four labeled primers have identical electrophoretic mobility. The stained conjugated primers retain their ability to specifically hybridize to DNA, as illustrated by their ability to replace the underivitized oligonucleotide normally used in sequencing reactions. End marking of DNA for use in the Maxam / Gilbert method.

In the Maxam / Gilbert method of DNA sequencing, the end of the piece of DNA whose sequence is to be determined must be marked. This is conventionally done enzymatically using radioactive nucleosides. To use the Maxam / Gilbert method in connection with the color detection scheme described in connection with this invention, the DNA fragment must be labeled with dyes. One way in which this can be done is shown in Figure 1. Some restriction endonucleases produce what is known as a 3 'overhang as the product of DNA cleavage. These enzymes provide a "sticky end", a short stretch of single-stranded DNA at the end of a piece of double-stranded DNA. This region will be attached (anneal) to a complementary stretch of DNA, which can be covalently joined to duplex DNA by the enzyme ligase. In this way, one of the strands is covalently bound to a traceable group. This group can be a dye, an amino group or a protected amino group (from which the protective function can be removed and which can react with dye after the chemical reactions).

Sequencing reactions.

The dideoxy sequencing reactions are performed in a standard manner according to Smith, A. J. H., Methods in Enzymology 65, 56-580 (1980), except that the scale may be increased if necessary to give an adequate signal intensity in each band for detection. The reactions are performed using a different color primer for each particular reaction, e.g. FITC for the "A" reaction, EITC for the "C" reaction, TMRITC for the "G" reaction and XRITC for the "T" reaction. No radiolabeled nucleoside triphosphate needs to be included in the sequencing reaction.

The Maxam / Gilbert sequencing reactions are performed in the usual manner, Gill, SF, Aldrichimica Acta 16 (3), 59-61 (1983), except that the end mark is either one or four colored dyes or a free or protected amino group, which can be reacted afterwards. taken with the dye.

Gel electrophoresis.

Samples from the sequencing reactions are combined and loaded onto a 5% polyacrylamide column 10 shown in Figure 2 from the upper container 12. The relative amounts from four different reactions in the mixture are adjusted empirically to give approximately the same fluorescence or absorptive signal intensity from each of the stained DNA conjugates. This enables compensation for differences in color extinction coefficients, color fluorescence quantum yields, detector sensitivity and so on. A high voltage is applied across column 10 to electrophorese the labeled DNA fragments through the gel. The labeled DNA segments, which differ in length by a single nucleotide, are separated by electrophoresis in this gel matrix. At or near the bottom of the gel column 10, the DNA bands dissolve from each other and pass through the detector 14 (described in more detail in the next section). The detector 14 detects the fluorescent or chromophoric bands of DNA in the gel and determines their color, and thereby which nucleotide they correspond to. This information provides the DNA sequence.

Detection.

There are many different ways to detect the labeled molecules, which are lengthwise separated using polyacrylamide gel electrophoresis. Four illustrative ways are described below.

These are i) detection of the fluorescence excited by light of different wavelengths for the different dyes, ii) detection of fluorescence excited by light of the same wavelength for the different dyes, iii) elution of the molecules from the gel and detection by chemiluminescence and iv) detection by absorption of light by the molecules. In methods (i) and (ii), the fluorescence detector shall meet the following requirements. (a) The excitation light beam shall not have a height significantly greater than the height of a band. This is normally in the range 0.1 to 0.5 m. The use of such a narrow excitation beam makes it possible to obtain maximum resolution of the bands. b) The excitation wavelength can be varied to match the absorption maxima for each of the different dyes or can be a single narrow, high-intensity light band that excites all four fluorophores and does not overlap any of the fluorescence emission. C) The optical configuration should minimize the flow of scattered and reflected excitation light to the photodetector l4. The optical filters, which block scattered and reflected excitation light, vary as the excitation wavelength varies. d) The photodetector 14 should have a relatively low level of interference and a good spectral response as well as a quantity efficiency within the whole emission range of the dyes (500 to 600 nm for the above-mentioned dyes). e) The optical system for collecting emitted fluorescence should have a high numerical aperture. This maximizes the fluorescence signal. Furthermore, the depth of field for the optics collection should include the full width of the column matrix.

Two illustrative fluorescence detection systems are shown in Figures 3 and 4. The system in Figure 3 is compatible with either single wavelength excitation or multiple wavelength excitation.

For single wavelength excitation, the filter F4 is one of four "band pass" filters centered at the emission wavelength peak of each of the dyes. This filter is switched at intervals of a few seconds to enable continuous monitoring of each of the four fluorophores. For multiple wave- L 456 348 ll longitudinal excitation, the optical elements F3 (excitation filter), DM (dichroic mirror) and F4 (barrier filter) are connected together. In this way, both the excitation light and the observed emission light are varied. The system in Figure 4 is a good device for single wavelength excitation. This system has the advantage that no moving parts are required and fluorescence from all four dyes can be monitored simultaneously and continuously. A third way (iii above) to detect is to elute the labeled molecules at the bottom of the gel, combining these with a chemiluminescent excitation agent, such as 1,2-dioxetanedione, Gill, SK, Aldrichimica Acta 16 (3), 59- 61 (1983): Mellbin, GJ, Liq. Chrome. 6 (9), 1603-1616 (1983), and flow the mixture directly into a detector, which can measure the emitted light at four separate wavelengths (this detector is similar to that shown in Figure 4 but does not require an excitation light source). The background signal in chemiluminescence is much lower than in fluorescence, which results in a higher signal to disturbance conditions and increased sensitivity. Finally, the measurement can be performed by measuring the light absorption (iv above). In this case, a light beam of variable wavelength is passed through the gel, and the decrease in radiation intensity due to light absorption of the labeled molecules is measured. By measuring the light absorption at different wavelengths corresponding to the absorption maximum for the four dyes, it is possible to determine which color molecule is present in the light path. A disadvantage of this type of measurement is that the absorption measurements themselves are less sensitive than fluorescence measurements.

The detection system described above is adapted to a computer 16. at each time interval examined, the computer 16 receives a signal proportional to the measured signal intensity at this time for each of the four colored marks.

This information tells which nucleotide terminates the DNA fragment of the particular length in the observation window at this time. The temporal sequence of colored bands yields the DNA sequence. Figure 6 shows the type of data obtained according to conventional methods as well as the type of data obtained by the improvements described according to the present invention.

The following examples are intended to illustrate the invention only.

EXAMPLE Figure 6 shows a block diagram of a DNA sequencer to be used with one dye at a time. The beam (4880 Å) from an argon ion laser 100 is directed into the polyacrylamide gel tube (sample) 102 by means of a beam guide 104. Fluorescence excited by the beam is collected using a low f-number lens 106, passed through a suitable set of optical filters 108 and 110 for to eliminate scattered excitation light and is detected using a photomultiplier tube (PMT) ll2. The signal is easily detected on a strip printer. The DNA sequencing reactions are performed using a fluorescein-labeled oligonucleotide primer. The peaks on the printer correspond to fragments of fluorescein-labeled DNA of varying lengths synthesized in the sequencing reactions and separated in the gel tube by electrophoresis. Each peak contains fluorescein on the order of 10_15 to 10-16 moles, which is approximately equal to the amount of DNA obtained per band in one equivalent. sequencing gel using radioisotope detection. This indicates that the fluorescent label was not removed or degraded from the oligonucleotide primer in the sequencing reactions. It also shows that the detection sensitivity is fully adequate to perform DNA sequence analysis in this way. a)

Claims (19)

456 348 PATENT REQUIREMENTS 1. A method for electrophoretic analysis of DNA fragments prepared by DNA sequencing methods, characterized in that labeled DNA fragments are provided with at least one chromophore or fluorophore prepared by sequencing chemistry and said fragments are detected when they are cleaved by electrophoresis. through a gel. A method of DNA sequencing according to the chain termination method according to claim 1, characterized in that a primer oligonucleotide labeled with a colored marker is used. 3. A method of DNA sequencing according to the chain termination method according to claim 1, characterized in that a primer oligonucleotide labeled with a fluorescent marker is used. 4. A method of DNA sequencing according to the chemical degradation method according to claim 1, characterized in that DNA molecules labeled with a colored marker are used. 5. A method of DNA sequencing according to the chemical degradation method according to claim 1, characterized in that DNA molecules labeled with a fluorescent marker are used. 6. A method of DNA sequencing according to claim 1, characterized in that a set of four chromophores or fluorophores is used to label said DNA fragments prepared according to the sequencing chemistry. 7. A method of DNA sequencing according to the chain termination method, characterized in that the primer oligonucleotide used in each of the four sequencing reactions A, C, G and T has a different colored marker attached to it and that 456 348 É W samples of the above sequencing reactions are combined and electrophoresed together on polyacrylamide gel and detected after their separation on the gel. 8. A method of DNA sequencing according to the chain termination method, characterized in that the primer oligonucleotide used in each of the four sequencing reactions A, C, G and T has a special fluorescent marker attached thereto and that samples of the above-mentioned sequencing reactions combined and electrophoresed together on polyacrylamide gel and detected after their separation on the gel. 9. A method of DNA sequencing according to the chemical degradation method, characterized in that the DNA molecules are labeled with different colored markers and that a different colored DNA is used in each of the chemical modification reactions and that samples of the above-mentioned the sequencing reactions are combined and electrophoresed together on a polyacrylamide gel and detected after their separation on the gel. 10. l0. Methods of DNA sequencing according to the chemical degradation method, characterized in that DNA molecules are labeled with different fluorescent markers and that different fluorescent DNAs were used in each of the chemical modification reactions and that samples of the above sequencing reactions are combined and electrophoresed together on a polyacrylamide gel and detected after their separation on the gel. 11. ll. Methods of DNA sequencing according to the chemical degradation method, characterized in that DNA molecules are provided with an amino group, which is linked to a color molecule after the sequencing reaction. 12. l2. DNA sequencing methods according to the chemical degradation method, characterized in that DNA molecules are provided with a protected amino group, which is unblocked and linked to a color molecule after the sequencing reactions. 456 348 I5 13. The method according to claim 11, characterized in that the products of each of the different sequencing reactions are coupled with a different colored dye, that samples of said color-labeled reaction products are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel. . 14. l4. The method according to claim 12, characterized in that the products of each of the different sequencing reactions are coupled with a different colored dye, that samples of the color-labeled reaction products are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel. Electrophoretic analysis of DNA fragments prepared by DNA sequencing methods comprising: establishing a source of chromophore- or fluorescently labeled DNA fragments from sequencing procedures, establishing a zone for containing an electrophoresis gel, establishing devices for introducing said labeled DNA fragments established in said zone, establishes photometric devices for monitoring said DNA fragments as they move through said gel, and monitors said labeled DNA fragments as they are introduced into said zone and moves through said gel. The new method according to claim 15, characterized in that the photometric device is an absorption photometer. 17. The new method according to claim 15, characterized in that the photometric device is a fluorescence photometer. The novel method according to claim 15, characterized in that the DNA fragments are labeled with an amino group linked to a color molecule. 456 348 W 19. The novel method according to claim 15, characterized in that a set of four chromophores or fluorophores is present - to label said DNA fragments from the sequencing method.