DE19937512B4 - Method and apparatus for rapid genome sequencing by linearization or separation of the DNA - Google Patents

Abstract

The invention relates to the field of molecular biology and genomics. Two principally different sequencing methods are used virtually invariably today. DOLLAR A The object of the invention is to achieve a faster and cheaper sequencing of genomes. The sequencing of a pro- or eukaryotic z. B. a human chromosome z. B. succeed within a month or a week and run as automatically as possible. DOLLAR A The object of the invention is achieved by a purified genomic DNA sequence or an unpurified DNA sequence of anaphase, telophase, G1-phase or mitosis or interphase chromosome decomposed in a mixture of proteases on a long plate ( LP), linearized on a ruler or on or along a long bar and then with such procedures such. B. laser scanning microscopy z. B. under weak UV irradiation (260-280 nm or 200-250 nm), which is absolubilized by the DNA, scanning tunneling microscopy, electron microscopy (EM), NMR or other spectroscopic analysis, CD (Circular Dichroism)) or sequenced by another method.

Description

The The invention relates to the field of molecular biology and genomics. Two fundamentally different sequencing methods are used today practically invariably applied.

The The first method is based on a base-specific chemical cleavage at the end of radioactively labeled DNA fragments (Maxam-Gilbert method). In 4 different reactions the DNA fragments on the 4 bases become partially base-specific modified. Thereafter, the modified base of the deoxyribose removed and the phosphodiester bond hydrolyzed at this point in the DNA backbone. The resulting fragments are then on very thin polyacrylamide gels separated in the electric field according to size. After autoradiography only the fragments are visible, which are still one possess intact end, since the DNA is only radioactively labeled there has been.

The second method, which is much more common today, is based on the enzymatic re-synthesis of the DNA to be sequenced in the presence of nucleotide-specific inhibitors (Sanger method). Starting from a short oligonucleotide (primer), a DNA strand is re-synthesized in 4 separate batches on the DNA to be sequenced. At the same time, the newly synthesized DNA is radiolabeled by incorporation of ³² P-dATP. However, each batch is added to one of 4 nucleotide-specific inhibitors in addition to the substrates (dNTPs) required for DNA synthesis. These are dideoxynucleoside triphosphates (ddNTPs), which lack not only the 2'-OH group but also the 3'-OH group of deoxyribose. Dideoxynucleoside triphosphates, as well as deoxynucleoside triphosphates, are used as substrates by the DNA polymerase. However, if they are randomly incorporated into the DNA instead of a deoxynucleoside triphosphate, then at this point the DNA chain can no longer be extended, since the 3'-OH group is missing. In each of the 4 reaction approaches, therefore, there is a population of DNA molecules that are all radiolabeled on the one hand, on the other hand all in the form of the primer have an identical 5 'end, and finally but in length to each distinguish base-specific 3 'end. These fragments are again size-separated on thin polyacrylamide gels in the electric field and displayed by autoradiography.

Of the The main disadvantage of these sequence analyzes is the long-term and financial expenses. From the beginning of the project to the point where A genome completely sequenced can take years and even decades pass, e.g. became the "Human Genome Project "im Started in 1973 and is expected to finish in 2003.

The DE 199 29 530 A1 of the inventor, filing date 28.06.1999, disclosure 04.01.2001, describes in the 7 and 11 to 14 and in claim 18, the simultaneous sequencing of multiple sections of a DNA molecule moved across two cylindrical bodies. According to claim 9 of this document, the DNA molecules can also be stored on a long plate.

The US 5 079 169 A shows in 9B the supervision of a DNA molecule deposited on a long plate. In 6C This document shows the pulling apart of the DNA molecule. The sequencing is carried out according to column 3, lines 62 to 65 of this document by microscopy.

The US 5,674,743 A describes in column 8, lines 6 to 9, the sequencing of linearly stretched DNA molecules by detectors.

The The object of the invention is a faster and cheaper sequencing of genomes. The sequencing of a pro- or eukaryotic Genome or human chromosomes should e.g. within a month or a week succeed and possible run automatically.

The The object of the invention is achieved in that a purified genomic DNA molecule or a unpurified DNA molecule anaphase, telophase, decomposed in a mixture of proteases, G1-phase or mitosis or interphase chromosome on a long Plate (LP), on a ruler or on or along a long Stabs laid straight or linearized or pulled apart and then with such methods as e.g. Laser scanning microscopy e.g. under low UV irradiation (260-280 nm or 200-250 nm), which is absorbed by the DNA, scanning tunneling microscopy, electron microscopy (EM), NMR or other spectroscopic analyzes, CD (Circular Dichroism) or with another physico-optical method is sequenced.

preparation

a) purification of the genomic, e.g. chromosomal DNA molecules

A DNA molecule in the form of a nucleus isolated chromosome, from the plasma of an isolated organelle, or a viral or a Bacterial DNA or DNA of other origin is separated by degradation of chromosomal proteins or organelle proteins from the proteins and then purified, for example by centrifugation.

Around to achieve a degradation of chromosomal proteins, at least a chromosome is incubated with a mixture of proteases. The mixture of Contains proteases e.g. Chymotrypsin, Streptomyces griseus protease (pronase), Aspergillus oryzae protease, subtilisin, elastase, pepsin, papain, bromealin et al Proteases and dithiothreitol, which disulfide bridges in Reduced proteins.

By this protease decomposes the chromosomal proteins, wherein the DNA molecule is not attacked and released. The DNA molecule is e.g. by centrifugation e.g. in cesium chloride Gradient or gel electrophoresis cleaned.

b) Preparation of the purified DNA molecule

For sequencing is an immobilization of the two ends of the genomic DNA molecule to certain Substrates necessary. These substrates may e.g. be square or circular, where a page or surface e.g. a rectangle or circle is connected to a glass rod and on the other surface a short synthetic DNA molecule, e.g. immobilized a poly (G / C) n molecule is. The quadrangular substrates can along a long plate (LP) or on the surface of the LP or over move a groove in the LP. The circular substrates can become also along the LP or over or move in the groove in the LP. The short synthetic DNA molecule may e.g. in a DNA synthesizer and either by a Oxygen atom or e.g. by polylysine-polyhistidine fusion peptides, which are mounted on a nickel-containing surface to which Substrates are immobilized. The LP has at least one depression which can be either small or as a test tube. The long disk (LP) consists e.g. made of glass, silicon or a metal such as. Gold or copper. The LP can be fixed or elastic or be flexible. The surface the LP can be good or precise or not honed well. On the LP can be one, two or more Move substrates that are over the level of the depression and the groove. The two free Ends of the immobilized on two substrates synthetic DNA molecules are located yourself in the depression, but are relatively far apart, to prevent the ligation between these free ends. The Immobilization of the genomic or chromosomal DNA molecule becomes by the ligation of one or both ends of this genomic DNA molecule with the free ends of the short synthetic DNA molecules e.g. Poly (G / C) n, respectively are immobilized at one end to the movable substrate, in the Deepening of the LP and achieved with the addition of DNA ligases and ATP.

c) Ligation of the two Terminals of the purified or unpurified genomic or chromosomal DNA molecules with the free ends of the synthetic DNA molecules of the substrates

The purified genomic e.g. chromosomal DNA molecule is in the depression in or on the surface of the long plate (LP) or her groove. Thereafter, a mixture with DNA ligases e.g. T4 ligases and ATP in this well, so the two ends of the purified genomic DNA molecule with the two free ends the synthetic DNA molecules such as. Poly (G / C) n of the substrate are ligated. In the depression may also be a G1-phase or an interphase or a mitotic chromatin be placed. In this case, you first give a mixture Proteases, e.g. Chymotrypsin, Streptomyces griseus protease, Aspergillus oryzae protease, subtilisin, elastase, pepsin, papain et al Proteases to allow the chromosomal proteins without impairment DNA and then a mixture with T4 ligases, ATP and inhibitors of proteases in the well on the LP.

you can be several mutually ligated chromosomes or chromosomal DNA molecules Insert into the well on the LP to allow sequencing of the entire genome can be uninterrupted.

For the ligation the chromosomes are taken e.g. G1-phase chromosomes and treated their Telomere sections so that the telomere areas become free. After that mix these chromosomes, whose telomere sections are free, with DNA ligases and ATP. Thereby the telomere sections become different Chromosomes ligated together. One introduces these ligated chromosomes in a mixture of proteases, after which the chromosomal proteins without impairment The DNA are decomposed and the DNA molecules uncovered or unscrewed become.

linearization or pulling apart

To the ligation of the two ends of the purified or unpurified genomic or chromosomal DNA molecules with the free ends of the short synthetic DNA molecules e.g. (G / C) n, which are each immobilized at one end to the movable substrates, These substrates are opposed on the long plate (LP) Directions slowly moved. The exposed DNA molecule will diverge drawn. This DNA molecule is on the surface of the Groove of the LP stretched or rectilinear or linearized.

sequencing

To the linearization of genomic or chromosomal DNA molecules on the long plate (LP) is a DNA molecule with methods such. Laser scanning, Electron or Scanning tunneling microscopy, NMR or other spectroscopic analyzes or other physico-optical methods.

The linearized DNA molecule can through multiple sequencers and also with different fragments be sequenced. This process is referred to herein as "multi-parallel sequencing" (MS). To simplify the sequencing relatively, one can see the surface of the LP or groove with a mixture of helicases, e.g. 3'-5 'E. coli Rep Helicase, and ATP coat so that the linearized double-stranded or dsDNA in two complementary single ssDNA molecules is shared. You can heat the whole LP or just the deepening, so that all hydrogen bonds and DNA-DNA interactions solved become.

In the 1 is an interphase eg G1-phase, a mitosis chromosome or anaphase chromatin 10 which is in a mixture of proteases 11 in the test tube 9 is placed, and pipette 20 with the purified genomic or chromosomal DNA molecule 7 shown schematically. The three arrows from the test tube 9 to the pipette 20 mean separation, purification of the molecule 7 and the introduction to the pipette 20 After the chromosomal proteins in the mixture of proteases 11 be demoted.

In the 2 is a long record (LP) 2 , and the depression 3 shown in the LP2.

In the 2a is the LP2 without groove and the recess 3 shown. The view A shows the LP2 from the side. The view B shows LP2 from above and the view C shows the LP2 from the front. The view D is a view of the LP2.

In the 2 B is the LP2 with the groove 5 and deepening 3 shown. The view A shows the LP2 with the groove 5 and the depression 2 from above. The view B is a view of the LP2 with the groove 5 but without the depression 3 , The view C also shows a room view of the LP2 with the groove 5 and the depression 3 ,

In the 2c is a long rod as a configuration of the LP2, with the groove 5 and the depression 3 shown from above (view A). The view B shows the groove 5 and the depression 3 in this rod schematic from the front.

In the 3a and 3b are circular and quadrilateral substrates 17 that with the glass rods 16 connected or mounted on these rods are shown.

In the 3a is a substrate 17 with the glass rod 16 and the synthetic DNA molecule 4 represented (view A). The view B1 shows a circular substrate 17 with the molecule immobilized in the middle 4 from underneath. The view B2 shows a quadrangular substrate 17 with the molecule immobilized in the middle 4 from underneath. The molecule 4 is by an oxygen atom on substrate 17 immobilized.

In the 3b is a substrate 17 Mounted on a rod and bottom with nickel-NTA or Ni-NTA (nickel-nitrilo-acetic acid) layer 15 is coated, and the molecule 4 shown schematically. The Ni-NTA layer 15 interacts with a layer of purified polyhistidine-polylysine fusion peptides 14 on which the molecule 4 is immobilized. Polyhistidine binds to nickel-NTA or Ni-NTA, and polylvsin binds to a DNA molecule (view A). So can on this substrate the synthetic molecule 4 be immobilized with one end. The other end remains free. View B1 shows a circular substrate 17 with the molecule immobilized in the middle 4 and the Ni-NTA layer from below. View B2 shows a quadrangular substrate 17 with the molecule immobilized in the middle 4 and the Ni-NTA layer from below.

In the 3c is a square substrate 6 , which is not attached to any rod, but can move along the LP2 shown. substratum 6 contains the Ni-NTA layer 15 and the polyhistidine-polylysine fusion peptide layer 14 at which the molecule 4 is immobilized. View A shows substrate 6 with the Ni-NTA layer 15 , the polyhistidine-polylysine fusion peptide layer 14 and the molecule 4 of the page. View B shows substrate 6 from above, view C shows substrate 6 from right or left, with the layers 14 and 15 and molecule 4 ,

In the 4 are substrates 6 with the Ni-NTA layers 15 and polyhistidine-polylysine fusion peptide layers 14 , the lids 13 the substrates 6 , the pipette 20 and the LP2 with the groove 5 in which a chromosome 10 or a molecule 7 lies that through the

Ligation sites 8th with the molecules 4 connected is. The arrows show the laying on of the lids 13 on the substrates 6 , Through the pipette 20 becomes the chromosome 20 or the molecule 7 on the groove 5 the LP2 laid.

In the 5a and b are substrates 6 extending in opposite directions along the LP2 with the groove 5 move, and the chromosome 10 or the molecule 7 pull apart or linearize, shown. The arrows show the movement movement of the substrates in opposite directions.

In the 6a and b are sequencers 1a , b and c and the LP2 shown. The arrows show the movement of the sequencer 1a , b and c. The DNA molecule linearized on the LP2 7 is sequenced in several sections simultaneously. This process is multiparallel sequencing (MS). The sequencers can move over the LP2, but also the LP2 can move under the sequencers.

In the 7a is the LP2 with the recess 3 of the molecule 7 and the heater 18 shown. The capacity of the well 3 is similar to the capacity of the test tube 3 , The heater 18 heats the depression 3 and hence the molecule 7 whereby hydrogen bonds and DNA-DNA interactions are broken in this sequence. The depression 3 is in the middle of the LP2.

In the 7b is the LP2 with the recess 3 , the molecule 7 and the heater 18 shown. On the LP2 is the substrate 6 with the molecule 4 whose free end is in the depression 3 located. After ligation of the one end of the molecule 7 with the free end of the molecule 4 in the depression 3 , this depression is through the heater 18 heated. This breaks down hydrogen bonds and DNA-DNA interactions and then becomes the substrate 6 which pulls the molecule out of the well and linearizes on the LP2. The depression 3 is on one end of the LP2.

In the 8th is the reaction of ligation of chromosomes 10 in the mixture of DNA ligases and ATP 12 shown schematically. The chromosomes are greatly enlarged. The telomeres are treated so that the telomere sections (TS) [engl. telomeric repeat sequences TRS] or their ends become free. In the mixture of DNA ligases and ATP 12 become the free ends of the chromosomes 10 ligated together. The ligated chromosomes 10 are then degraded, for example in the well or in the groove of the LP by proteases and the DNA molecule is linearized or pulled apart.

Consequently can e.g. linearized three or more chromosomal DNA molecules on a LP and be sequenced.

An anaphase, telophase, G1-phase or mitosis or interphase chromosome 10 , which is shown schematically in the 1 is treated so that the chromosomal proteins are degraded, whereby the chromosomal DNA molecule is not impaired, but unfolds and unscrews. Thereafter, this DNA molecule is separated or isolated, purified and then placed in a pipette 20 introduced. Isolation of DNA from a cell may be accomplished, for example, by centrifugation, or a cell may be taken, for example, a white blood cell (a leukocyte) and placed in a mixture of CHAPS / CHAPSO, which dissolves lipid membranes without denaturing proteins, sodium dodecylsulfate, which Lysis of cell nuclei causes proteinase K, which degrades the nucleosomes, including proteases and dithiothreitol, which reduces disulfide bonds in proteins. The cell is decomposed and the DNA is isolated from the cell fluid and added to the pipette 20 introduced. In the pipette 20 are not the chromosome (s) 10 but the genomic or chromosomal DNA molecules 7 ,

The long plate (LP) 2 in the 2 indicates the depression 3 on, in which the molecule 7 through the pipette 20 is introduced or introduced.

The molecule 7 in the depression 3 must be linearized on the LP2 and then through multiple sequencers 1 (a, b and c) are sequenced. The linearization of the molecule 7 is achieved by the fact that the two ends of this molecule 7 with the free ends of the molecules 4 be ligated.

The synthetic molecules 4 in the 3 are at one end or square or circular substrates 17 or 6 immobilized. The other end remains free. Immobilization of one end of the molecule 4 to a particular substrate 17 or 6 is achieved, for example, by polyhistidine-polylysine fusion peptides on a nickel-containing surface. Polyhistidine eg His _{(10 As)} binds to nickel-containing substances such as Ni-NTA (nickel-nitriloacetic acid), and polylysine eg Lys _{(10 As)} binds to the DNA.

The substrate 6 in the 4 contains a Ni-NTA layer 15 and a polyhistidine-polylysine fusion peptide layer 14 at which the molecule 4 is immobilized with one end and the other free end is in the groove 5 the long plate (LP) 2 ,

The substrate 6 will with the lid 13 covered to the immobilized molecule 4 to fix again. The two substrates 6 with the lid 13 ( 4 ) can move in opposite directions on the LP2. Before the substrates 6 begin to move in opposite directions becomes either a chromosome or chromatin 10 or a purified chromosomal or genomic molecule 7 in the groove 5 in the space between the substrate 6 by means of the pipette 20 placed. Thereafter, a mixture of proteases or a mixture of proteinase K and dithiothreitol is added to the chromosome 10 which is in the groove 5 the LP2 is located to decompose the chromosomal proteins without affecting the DNA. Then you give a Ge containing inhibitors of the proteases to inhibit them, DNA ligases and ATP into the groove 5 to the decomposed chromosome 10 , This will be the two ends of the chromosome DNA 10 / 7 with the free ends of the substrates 6 immobilized DNA molecules 4 ligated. If you have a purified genomic or chromosomal molecule 7 on the groove 5 between the substrates 6 through the pipette 20 hangs up, so you only give the mixture of DNA ligases and ATP to the molecule 7 so that the two ends of this molecule 7 with the free ends of the molecules 4 be ligated. After ligation of the two ends of the molecule 7 with the free ends of the molecules 4 catch the substrates 6 to move in opposite directions on the LP2 ( 5 ). Through this movement of the substrates 6 in opposite directions, the chromosomal or genomic molecule is pulled apart so that the farther the substrates from each other, the more straightforward the molecule becomes 7 becomes. The molecule 7 becomes linear, and this process is called "linearization" of the DNA molecule 7 '' designated. The substrates 6 move slowly, but to speed up the linearization can be the LP2, the groove 5 or just the depression 3 heated, eg, to 90 ° C, or heated, allowing hydrogen bonding and DNA-DNA interactions of the nanomass of the molecule 7 to get broken.

After linearization of the genomic or chromosomal DNA molecule 7 It will be through one or more sequencers 1 sequenced ( 6 ). The linearized molecule 7 can be sequenced in several areas at the same time. This process is called multiparallel sequencing of the linearized DNA molecule 7 , The DNA molecule linearized on the LP2 7 may be a double helix (dsDNA), or separated into two single-stranded molecules (ssDNA) by heating or helicases, for example. The one or two complementary ssDNA molecules can be more accurately sequenced.

The LP2 in the 7 has a recess 3 on, its capacity to the test tube 9 is similar. This depression and, consequently, its contents will pass through the heater 18 heated, breaking hydrogen bonds and DNA-DNA interactions.

However, heating occurs after ligation of either end of the molecule 7 with the free end of the molecules 4 , The other end of the molecules 4 is to the substrate 6 immobilized. So that's the depression 3 through the heater 18 after ligation of the one end of the molecule 7 with the end of the molecule 4 heated. The DNA-DNA interactions and the hydrogen bonds are broken and the substrate 6 ( 7b ) begins; to move. By the movement of the substrate 6 becomes the DNA molecule 7 from the depression 3 drawn and linearized on the LP2. The sequencing of each chromosome can be done in parallel on different LP2, or you can have multiple chromosomes 10 or DNA molecules 7 ligate each other to allow linearization and sequencing of multiple chromosomal DNAs 7 / 10 can be done on a LP.

In the 8th is the process of ligation of multiple chromosomes 10 by DNA ligases and ATP in the mixture 12 shown schematically. To achieve the ligation, the telomeres are treated to expose telomere sections LTS (or telomeric repeat sequences TRS). The ends of these telomere sections will also become free and if you place multiple chromosomes with free telomere ends in the mixture 12 of DNA ligases eg T4 ligases and ATP, the free telomeres and consequently the chromosomes are ligated together.

1

sequencer, e.g. Probe a raster tunnel microscope or a

spectroscope

2

Long Plate (LP)

3

deepening

4

(Short) synthetic DNA molecules

5

groove on the LP2

6

angular substrate without glass rod 16

7

one genomic or chromosomal DNA molecule

8th

Ligation site between the molecule 4 and the molecule 7

9

Test tube

10

one Interphase, e.g. G1 phase, or mitosis chromosome or anaphase

chromatin

11

mixture with proteases

12

mixture with DNA ligases and ATP

13

Cover of the substrate 6

14

Polyhistidine fusion peptide-polylysine-layer

15

Ni-NTA-layer 16 glass rod

17

substratum of the glass rod

18

heaters

19

Connecting fiber between the glass rod 16 and the role 18

20

pipette

Claims (2)

A method for rapid genome sequencing, characterized in that more than two regions of a linearized or stretched DNA molecule placed on a flat long plate are simultaneously, multiparallel, sequenced by physico-optical methods, wherein the sequence to be sequenced, linearized or stretched DNA molecule is provided, by coupling both of its ends to substrates via synthetic DNA molecules, and simultaneously moving these substrates in opposite directions for purposes of linearizing or pulling apart the DNA molecule. Apparatus for carrying out the method according to claim 1.