DE102008037890B3 - Sequence analysis of nucleic acids e.g. DNA, comprises isolating and arranging, depositing nucleic acid on support, unwinding nucleic acid and determining unwinding of nucleic acid vibrations, over assigned vibrations of support - Google Patents

Abstract

Sequence analysis of nucleic acids, comprises: (a) isolating and spatially arranging, orderly placing or depositing a nucleic acid on a support (1), (b) unwinding the nucleic acid and (c) determining the unwinding of the nucleic acid vibrations, over the assigned vibrations of the support by e.g. laser beam reflection analysis, ion beam reflection analysis, electron beam reflection analysis, field modification analysis, voltage change analysis, potential change analysis between the oscillating support and a neighboring surface, and/or analysis of the current flow change by the support. Sequence analysis of nucleic acids, comprises: (a) isolating and spatially arranging, orderly placing or depositing a nucleic acid on a support (1), (b) unwinding the nucleic acid and (c) determining the unwinding of the nucleic acid vibrations, over the assigned vibrations of the support by laser beam reflection analysis, ion beam reflection analysis, electron beam reflection analysis, field modification analysis, voltage change analysis, potential change analysis between the oscillating support and a neighboring surface, analysis of the current flow change by the support, by electromagnetic detection procedure, microscopy or atomic force microscopy, optical interference microscopy, scanning tunnel microscopy, optical-fiber interferometer, spectral analysis, by detecting mechanical vibrations, and/or by thermosensoric procedures, as a single procedure or in combination, where the nucleic acid is sequenced by vibration analysis. An independent claim is included for a device for performing the method, where: at least one support is adapted for at least one nucleic acid; a receiving- or recording-device for vibration of the support is formed; and the receiving of vibrations is based on the measurement method according to step (c), individually or in combination.

Description

The The present invention includes novel sequencing methods for the determination the base sequence of nucleic acids, DNA or RNA. Once a single nucleic acid is spatially arranged and the conditions in the cell are mimicked in vitro, the DNA controlled or ordered unwound in vitro or replicated will, can the vibrations resulting from the DNA unwinding in vitro for the Sequence determination can be used.

The Sources of these vibrations are z. B. DNA unwinding, DNA replication, DNA transcription, as well as DNA rewinding or rewinding. Once the DNA double helix escapes, beat the struggling single strands "the ground" or the Carrier, they oscillate and fluctuate the medium. Once it's a the only in vitro DNA sequence to be determined and the unwinding in a certain unwinding direction, you can with different known methods record these oscillations or vibrations or detect or measure and assign to the respective nucleotides or determine the order of the nucleotides, d. H. sequence. Each stroke is different and dependent on the parallel or antiparallel sequence, the breaks between A and T (2 hydrogen bonds) and G and C (3 hydrogen bonds), from the position of the base pairs in the overall sequence and from the Length of total sequence, torque and the absence of other similar vibrations or signals to prevent signal mixing as much as possible. This lends itself to the vibrations of a single or single sequence to determine. The parallel sequence is in the unwinding direction always a phosphate heavier, as the antiparallel sequence beats too stronger than the antiparallel sequence. The shock or energy release at the break of the hydrogen bond between G and C is stronger as when breaking the hydrogen bond between A and T.

The The object of the present invention is therefore the DNA sequence by unwinding in vitro. This method can be called DNA sequencing by in vitro unwinding or Sequencing by Frequencing or SeqbyFreq or SeqFreq.

The closest prior art are the DE 199 29 530 A1 and the DE 199 37 512 A1 with the spread of DNA single strands.

The EP 0 533 302 A1 describes an infrared DNA sequencing.

The EP 1 574 837 A1 shows in 2 tunneling DNA sequencing of a single DNA molecule, with strand separation provided.

MATSUMOTO, A. & OLSON, W. K. [Sequence Dependent Motions of DNA: A Normal Mode Analysis at the base-pair level. Biophys. J. (2002) 83 (1) 22-41] Measurements of DNA dynamics at the base level.

ADAIR, R.K. [Vibrational Resonances in Biological Systems at Microwave Frequencies. Biophys. J. (2002) 82 (3), 1147-1152] and GOLD, V.L. [Three-wave interaction between interstrand modes of the DNA. Journal of Experimental and Theoretical Physics (2005) 101 (2) 372-379] examine DNA responses in the microwave frequency range.

None However, prior art documents teach the process of unwinding or rewinding to benefit from Helices, to detect vibrations of the bases.

The The object of the invention is achieved by a method and a device according to this Invention solved. A single, preferably chromosomal DNA sequence is isolated, z. B. on a surface or spatially on a plate arranged and by the addition of ATP and DNA helicases or by the addition of alcohol, Alkali or acids or controlled by gradual heating controlled. It should it is best to bind or immobilize one end of the DNA sequence to be determined and the other end should be left free to allow the unwinding the DNA sequence runs in a certain unwinding direction d. H. starting from the free end and continuously bound to the other The End. The other bound end thus prevents by his bondage the start of the unwinding from the bound end. The bound end can also rotate or rotate or rotate with the DNA. Ie. the Unwinding begins only at the free end and moves in the unwinding direction bound end continued. This will give a clearly defined or definable Oscillation reaches that of the escaping DNA sequence on the surface or plate on which this DNA sequence is located and this plate or platform then oscillates accordingly the DNA sequence oscillating through the unwinding. The oscillations or vibrations of this plate can vary with different Procedure to be determined. These methods include the reflection analyzes of the reflecting laser, ion, or electron beam, analysis dynamic field, voltage, or potential changes between the vibrating plate and another obvious surface, atomic force microscopy, Optofaser interferometry and optical interference microscopy.

The Entwindungsgeschwindigkeit, d. H. the number of separate from each other Base pairs per unit time (s) or (ms) depends on the intensity of the effect of the vanishing mechanisms. The determination or sequencing speed depends accordingly from the unwinding rate of the DNA sequence. If it is to simple Entwindungsmechanismen such. B. by alcohols, acids or Bases acts, hangs the unwinding rate of the denaturing concentration Substances, d. H. the more concentrated or the more denaturing Substances are present, the greater the Entwindungs- and according to the sequencing speed. Ie. you can get the sequencing speed and unwinding rate by concentrations, pH and / or regulate by temperature.

The Disengaging DNA in itself, by its unwinding the source of mechanical vibrations. These vibrations are different or dependent from the hydrogen bonds (2 bridge bonds between A and T) and 3 bridge bonds between G and C).

The Unwinding the double helices (Helices) gets punches and mechanical Oscillations, d. H. strands hit the wearer or the plate or the surface, on which the process takes place. As already mentioned, will be these oscillations z. B. with laser beam reflection analysis, atomic force microscopy, Voltage change analysis recorded and the obtained data are interpreted d. H. translated into the order of nucleotides. The interpretation is based on the following theoretical basis: the recorded and stored oscillations or oscillations represent a curve, where corresponding peaks for minimum and maximum beats or Strength these blows are. The peak analysis also includes the analysis of the distances between minimum and subsequent maximum peaks, since these distances are base-pair specific. The steps of the curve are alternately on different Height, d. H. in different areas.

Of the an area is for the so-called PER sequence (PER = parallel to the unwinding direction) and the other area is for the so-called APER sequence (APER = antiparallel to the unwinding direction). Because the strands are antiparallel, beats the PER sequence or the PER strand stronger than the APER sequence or the APER strand. Ie. the 3'-5 'sequence winds up stronger as the 5'-3 'sequence, because of the Beginning of the unwinding in the unwinding direction and then following in Entwindungsrichtung always has a phosphate group more or around this Phosphate is heavier and therefore more in the unwinding carrier suggests. This fluid "interval asymmetry" or "period asymmetry" of the unwinding allows it, specifically the PER sequence to "listener" or sequence.

• a) Isolierung und räumliche Anordnung oder geordnetes Legen oder Ablegen einer Nukleinsäure auf einem Träger,
• b) Entwindung der Nukleinsäure,
• c) Bestimmung der Entwindungs-Schwingungen der Nukleinsäure – über die zugeordneten Schwingungen des Trägers mittels Laserstrahl-Reflexionsanalyse, Ionenstrahl-Reflexionsanalyse, Elektronenstrahl-Reflexionsanalyse, oder/und – mittels Feldänderungsanalyse, Spannungsänderungsanalyse, Potentialänderungsanalyse zwischen dem schwingenden Träger und einer Nachbaroberfläche, oder/und – mittels Analyse der Stromflussänderung durch den Träger, oder/und – durch elektromagnetische Nachweisverfahren, Mikroskopie oder Atomkraftmikroskopie, optische Interferenzmikroskopie, Rastertunnelmikroskopie, Optofaserinterferometrie, Spektralanalyse, und/oder – durch Detektion mechanischer Oszillationen, und/oder – durch thermosensorische Verfahren, als einzelne Verfahren oder in Kombination miteinander, wobei die Nukleinsäure durch Schwingungsanalyse sequenziert wird.

The method according to the invention can thus be divided into the following steps:

• a) Isolation and spatial arrangement or ordered laying or depositing of a nucleic acid on a support,
• b) unwinding of the nucleic acid,
• c) Determination of the unwinding oscillations of the nucleic acid - via the associated vibrations of the carrier by means of laser beam reflection analysis, ion beam reflection analysis, electron beam reflection analysis, and / or - by field change analysis, voltage change analysis, potential change analysis between the vibrating support and a neighboring surface, and / or By analysis of the current flow change by the carrier, and / or by electromagnetic detection methods, microscopy or atomic force microscopy, optical interference microscopy, scanning tunneling microscopy, optofaser interferometry, spectral analysis, and / or by detection of mechanical oscillations, and / or by thermosensory methods, as individual methods or in combination with each other, wherein the nucleic acid is sequenced by vibrational analysis.

In a further embodiment of the invention, the Entwundene nucleic acid, preferably DNA, in step b) controlled orderly back wound and in this case the nucleic acid with the method of claim 1c) sequenced by vibration analysis become.

In a still further embodiment The invention may in step b) a unwinding and rewinding in the context of a replication of the nucleic acid, preferably DNA, run off and here the nucleic acid with the method of claim 1c) sequenced by vibration analysis become.

DNA isolation is known (see DE 199 29 530 A1 and DE 199 37 512 A1 ). The spatial arrangement is preferably carried out by adhesion and cohesion forces of the water, the buffer or the solution, as well as by magnetic forces, a DNA sequence in a straight line on a support or on a disk, spirally on a disk or on a groove on a disk , wave in a groove on a plate. The magnet can be either on the side or under the carrier. The deposit process takes place for. B. by pipette or micropipette. The in vitro DNA unwinding, as mentioned above, by addition of DNA-He Liquors and ATP or alternatively by alcohols, methanol, ethanol, mercaptoethanol and by bases, acids or heating.

The In vitro DNA replication occurs by adding DNA helicase, ATP, other enzymes and proteins of the DNA replication machine such as DNA polymerase, DNA primase, transcription factors and stabilizing proteins, which were previously prepared and purified recombinantly. The in vitro sDNA recovery or in vitro single-stranded DNA recovery is done by controlled gradual merging of DNA single strands (single chains) on the surface a carrier.

The DNA single strands can separated from each other by a moving partition or needle become. This partition prevents the uncontrolled Rückwindung or recovery of the single strands.

The slow movement of the partition allows the controlled step by step or continuous removal of the delimitation and thus allows the gradual return or recovery of DNA single strands. Through this recovery There are also vibrations or oscillations caused by calorimetry and can be determined by receiving the mechanical vibrations.

Of the Reception or recording of mechanical vibrations takes place as already mentioned, by Atomic force microscopy, interference analysis, spectral analysis, laser beam reflection change analysis, Electron and ion beam change analysis, Current, voltage, field change analysis or by other methods.

The Interpretation is done by the curve analysis with a special Computer program, wherein in the peak analysis, the respective peaks assigned to specific areas, d. H. in the PER sequence area or in the APER sequence area.

In the 1 Carriers for nucleic acids are shown schematically. The 1a shows view A or top view. The carrier 1 in the 1a contains groove or recess 2 with medium for DNA 23 and DNA sequence stretched in this medium 4 , The 1b shows the view B or the side view. The 1c shows the view C and you can see the depression in a transverse step. In the 1b and 1c is the DNA sequence 4 in the medium 23 shown schematically.

In the 2 Carriers are also shown schematically. The carrier 1 in the 2a is shown from above and shows the depression 2 with the stretched DNA sequence 4 , At the top of the needle-shaped support 1 is the mirror field 28 shown for the laser beam reflection analysis. The carrier 1 is with the substrate 13 connected. In the 2 B is the carrier 1 shown in cross section. This carrier 1 contains the DNA sequence 4 in the medium 23 in the depression 2 ,

In the 3 also carriers are shown which are intended for immobilized DNA sequences. The carriers 1 in the 3a contains the mirror field 28 and the depression 2 with the stretched DNA sequence 4 , This DNA sequence 4 is with the dsDNA fragment 14 ligated for ligation and thereby to the Immobilisierungsstelle 27 bound.

The immobilization site 27 in the 3b is a needle with the ability to rotate.

In 4 is the pipette or micropipette 25 when laying or placing the DNA sequence 4 into the depression 2 the carrier 1 shown schematically.

The DNA is in the medium after depositing 23 in the depression 2 , The DNA sequence emerges from the opening at the tip of the micropipette 25 with the help of the rotating magnet 26 ,

In the 5 Different carriers are shown schematically.

The carrier 1 in the 5a contains the recess 2 with stretched DNA sequence 4 , as well as the outstanding mirror field 28 ,

The carrier 1 in the 5b is by lever or outrigger 31 with the substrate 13 connected.

The carrier 1 in the 5c is also by lever 31 with the substrate 13 connected, but still contains prominent mirror field 28 ,

The carrier 1 with the recess 2 in the 5d is shown in cross-section and contains the superconducting layer 30 ,

The carrier in the 5e consists of airgel 33 with carbon or diamond coating 32 ,

The carrier in the 5f consists of a superconductor 30 , the airgel 33 with carbon coating 32 ,

The carriers 1 in the 5g and 5h contain wells 2 with the DNA 4 and consist of senior ( 9 ) and non-conductive ( 10 ) Layers, the electrical conductor 9 in the 5g to at the top or to the upper part of the carrier goes out and the electrical conductor 9 in the 5h to the lower part of the carrier.

The carrier 1 in the 5i contains gel edge 29 ,

In the 6 Carriers are shown with wavy and spiral grooves for nucleic acid sequences.

The carrier 1 in the 6a contains the wave-shaped groove 2 with the corresponding wavy deposited or arranged DNA sequence 4 ,

In the 6b is a disk or disk carrier 34 with the spiral groove 2 for the DNA sequence 4 , shown schematically.

The disc 34 in the 6c is similar to the one in the 6b additionally contains the handle or lever or the arm 31 ,

In 7 the controlled process of rewinding or rewinding the DNA single strands (single chain DNA) in vitro is shown schematically. The DNA single strands 5 squirm back together while the needle or the divider 6 moves, thereby enabling gradual, free recovery. The double helix DNA sequence resulting from the rewinding or rewinding process 4 is in the depression 2 of the carrier 1 , where the partition 6 also in the depression 2 is located and moved.

In 8th the device and the process of vibrational analysis or sequencing by vibrational analysis of the DNA is shown in vitro.

The vibrations of the DNA sequence 4 in the depression 2 cause the vibrations of the vehicle 1 or be on the carrier 1 transfer. The carrier 1 is located and oscillates on the elastic body such. B. made of Resilin or Resilinkörper which is on the substrate 13 located. The vibrations of the wearer 1 and consequently the DNA sequence 4 on the carrier 1 are determined by laser beam reflection analysis. At the same time, the laser beam becomes 18 from the laser beam source 19 sent out and reflected from the carrier 1 into the laser beam receiving or analyzing system 20 ,

In the 9 For example, configurations of the "core" of the DNA sequencing device are shown in vitro by vibrational analysis.

In the 9a is the long lever-type carrier 1 shown. This contains the DNA sequence to be determined 4 in the depression or groove 2 and is connected to the substrate 13 , The carrier 1 has the ability to spring, as an end to the substrate 13 is attached and the other end remains free. This free end reflects the laser beam as it springs through the DNA vibrations 18 , This laser beam 18 runs from its source 19 and, after reflection, enter the analysis system 20 , This analysis system is z. As a CD camera or a system with multiple photodetectors.

The system in the 9b is similar to the one in the 9a , wherein the free end is on the elastic body or elastic Resilinkörper 22 springs or oscillates.

The system for in vitro sequencing by vibration analysis, which is incorporated in the 10 imaged analyzes vibrations based on the field change analysis.

In the system in the 10a the vibrations of the free end cause the field or voltage changes. These changes are made by the evaluation system 7 evaluated. The current flows over the line 17 and about the carrier 1 and at the free end of the vehicle 1 with the recess 2 with the DNA 4 arise fluctuating fields or vibrations of the free end of the carrier.

The carrier in 10b is structured in layers and the current does not flow through the whole carrier but through its conductive layer 9 , This conductive layer 9 is located between the non-conductive layers 10 of the carrier 1 ,

In the 11a is the scheme of vibration determination with the help of atomic force microscope 35 displayed. The boom contains the top 8th ,

The carrier 1 in the 11b contains the tip at the free end 8th over the surface such. B. the over gold surface 36 for measuring the electrical field and voltage changes as well as the electron emission or the electron flow from the tip. This "electron specification" is dependent on the strength of the vibrations of the carrier or the tip or respectively dependent on the strength or intensity of these vibrations. The vibration data are then in the evaluation system 7 evaluated.

The system in the 12a determines field changes due to vibrations of the wearer 1 , which has one end on the substrate 13 is attached and the recess 2 with the DNA sequence 4 contains. The free end of the vehicle 1 vibrates over the surface 36 , the power from and to the evaluation system 7 passes. Between the surface 36 and the free swinging end of the vehicle 1 there are the field or field changes made by the evaluation system 7 be evaluated.

The system in the 12b also analyzes vibrations of the carrier due to current changes. The arrows show current flows.

The current flows from the tips 8th off to the surface 36 , The electron beam 21 runs from a peak 8th over the ladder 9 or the superconductor 30 over the other tip 8th to the surface 36 and the changes are evaluated by the evaluation system. The leader 9 or the superconductor 30 with the top 8th are parts of the carrier 1 and vibrate with. Consequently, vibration changes correspond to the flow of current and field changes.

The carrier 1 in the 13 consists of two sub-carriers 1 , The carrier 1 with the recess 2 and DNA 4 is connected to and is located on the superconductor 30 and hovers over the other superconductor 30 on the other carrier 1 , which with the substrate 13 connected or attached to him. The DNA-associated oscillations are transmitted to the suspended carrier and are determined by laser beam reflection analysis. The laser beam 18 from the source 19 , reflects and enters the analysis system 20 with several photodetectors.

The system in the 14a analyzes mechanical vibrations of the substrate 1 by interference analysis or interferometric. The interference takes place in the system 16 with two forked light guides.

The laser light or light comes from the light source 11 , passes over the light guide 15 to the interference system 16 and the interference system 16 over the other light guide 15 to the photodetector 12 ,

In the system in the 14b Vibration changes are measured or determined by changes in light. The light comes from the light source 11 and passes over the light guide 15 to the vibrating carrier 1 and then over the other fiber 15 to the photodetector 12 , which is sensitive to intensity changes of the light.

The system in the 15 uses laser beams to analyze the vibrations of the in vitro DNA in several places simultaneously. Here is the DNA sequence 4 spirally on a spiral but mostly planar surface of the carrier 1 arranged.

The system in the 16a contains two carriers 1 one to the substrate 13 fastened and an exchangeable carrier for easy manipulation or handling. Both carriers are determined by laser beam reflection analysis method. The system in the 16b is similar to the system in the 16a , wherein the interchangeable carrier 1 on the elastic Resilinkörper 22 relieves or vibrates.

The 17 show carriers with discs 34 with the spirally deposited DNA sequence 1 , The carrier in the 17a contains three measuring bodies 39 , in the 17b two measuring bodies 39 and in the 17 no measuring body 39 ,

The cone-shaped carrier in the 18 carries the diagonally arranged DNA sequence 4 which is immobilized at the top with one end and the other bottom end is free. The cone-shaped carrier 1 is on the substrate 13 and is from the groove 2 surround. The cone carrier can be hollow.

The vibrations of the cone or cone-shaped carrier 1 are determined by laser beam reflection analysis.

The carrier 1 in the 19 moves in the substrate 13 and during this movement the denaturing medium rises 24 and escapes the DNA sequence 4 ,

In the 20a and 20b is the carrier 1 each arranged at different slopes or angles and the substrate 13 attached and the laser beam reflection analysis takes place in the 20a close to the free end of the DNA sequence 4 and in the 20b close to the bound or immobilized end of the DNA sequence 4 ,

The DNA sequence 4 will be in the 21 Unwrapped by thermal energy of one laser beam and with the other laser beam vibrations of the wearer are measured.

The DNA sequence 4 in the 22 is located in the electric field and conducts electricity through the carrier 1 flows. The electric current affects the Entwindungsverhalten the DNA and the vibrations generated thereby.

The DNA sequence 4 in the 23 is located and escapes and swings directly on the elastic body 22 , z. B. on the Resilinmasse, which are on the substrate 13 located.

In the 24 are carriers 1 shown using strings with substrates 13 are connected. The vibrations of the carriers are measured more sensitively on the strings.

In the 24a is the DNA sequence 4 perpendicular to the strings 37 sent to the sub strate 13 are bound.

The DNA sequence 4 in the 24b is also perpendicular to the strings 37 arranged, with the strings are arranged diagonally.

The DNA sequence 4 in the 24c is parallel to the strings 37 arranged.

The DNA sequence 4 in the 24d is also parallel to the strings 37 , arranged with the strings arranged diagonally.

The carrier 1 in the 25 is through double helix strings 38 to the substrates 13 bound.

The carrier 1 in the 26a is through string 37 between two substrates 13 tied and is also attached to the string 37 attached.

The vibrations of the wearer 1 are measured by laser beam reflection analysis. The beam 18 from laser beam source 18 reflected from the vehicle 1 and enters the laser beam reflection analysis system 20 ,

In the 26b is the carrier 1 shown in cross section. The carrier 1 is with the lower part of the string 37 attached and contains the groove or recess 2 for DNA sequence 4 in the medium 23 ,

In the 27 is the core of the device for unwinding and sequencing the DNA sequence 4 shown schematically.

DNA sequence 4 is immobilized with one end and located on the support 1 , which is with the tub or with the container 43 connected is. This container 43 contains denaturing medium 24 , such as For example, ethanol. The denaturing medium 24 can either from the laser heating system or from the microwave generator 40 to be heated. The denaturing medium rises 24 and forces the DNA sequence 4 for unwinding. This unwinding causes vibrations of the wearer 1 , which by laser beam 18 or measured or recorded or detected using the laser beam reflection analysis.

Alternatively, the denaturing medium 24 by adding it from the pipette or micropipette 25 climb.

The systems in the 28a and 28b are similar to the system in the 27 and show the dynamics of the regulated slope of the medium 24 and the movement of the laser beam reflection analysis system in the unwinding direction of the DNA double helix.

The denaturing medium 24 rises in the 28a by adding the new medium 24 and in the 28b by continuous heating by microwaves or laser heating system 40 ,

The carrier 1 in the 29 is in the container 43 and swings over or on the medium 42 for the wearer. The medium 42 can be water, oil or lubricant.

The DNA sequence 4 is how it is in the 30 respectively. 30a to 30e is shown on the ice 41 ,

The DNA sequence 4 escapes and swings on the ice 41 on the substrate 13 in the 30a , The vibrations of the ice 41 are recorded with the laser beam reflection analysis system.

The system in the 30b is similar to the one in the 30a where the substrate 13 contains the edge.

The system in the 30c is similar to the one in the 30b , where ice cream 41 edge 3 contains and the DNA sequence 4 is in the medium 23 for the DNA. The system in the 30d is similar to the one in the 30c , where ice cream 41 with the laser beam reflection analysis system 40 can be heated.

The system in the 30e is similar to the one in the 30d , where ice cream 41 through electrodes 45 with the electric or electronic heating system 44 connected is. The figures thus show different embodiments and modifications of the methods and devices or devices for sequencing by the vibration analysis.

All potential versions and modifications of these methods and apparatus according to this invention are made by this patent application and claims covered or covered.

The mentioned versions and restrict modifications Do not look at the ones described in the registration text and with figures are shown.

The Combinations of the inventive features of the present invention give new inventive features, such. B. the boom with the top for atomic force microscopy becomes a carrier itself the DNA sequence.

The optical laser beam reflection and interference based vibration detection method in their entirety with carriers of DNA give rise to new inventive Steps and features.

The figures show, in summary, carrier, device and method or processes of sequencing by vibration detection or recording. The 14a and 15b show integrated interferometric and intensity measurement based detection or vibration determination methods.

The 8th . 9a . 9b . 13 . 15 . 16a . 16b . 18 . 20a . 20b . 21 . 23 . 26a . 26b . 27 . 28a . 29 . 30a . 30b . 30c . 30d and 30e show determination of DNA vibrations by laser beam reflection analysis.

The 10a . 10b . 12a and 12b show the determination of DNA oscillations by field change analysis.

The 11a and 11b show the determination of DNA vibrations by atomic force microscopy.

The to be determined nucleic acid sequences restrict not only on the DNA. It can also be RNA double helices with RNA polymerases and reverses Transcriptases are treated or prepared.

The single strands The DNA and RNA can be mechanical be separated from each other nanotechnologically. The analysis can also with the help of ion beams and other rays and those reflecting changes as well as calorimetrically done. The carriers can be made of resilin, elastin, Collagen, keratin, polyethylene or another substance or Material exist.

The Advantages of the present invention are that long DNA sequences, whole chromosomal DNA sequences as well as genomes fast, and indeed within hours to a few days and cheap, very inexpensive in the Thousands or hundreds can be sequenced.

embodiment 1

Please refer 9a , The carrier with the purified, prepared and spatially arranged DNA sequence is bound to the substrate so that one end is attached to the substrate and the other end of the carrier remains free. The DNA sequence is stretched or linearly arranged and is located in the medium which is located in the well of the carrier. DNA helicases and ATP are added to the DNA sequence medium. As a result, the unwinding of the DNA single strands is induced. The resulting DNA sequence oscillates or oscillates. These vibrations occur through impacts of the separating single strands of the evolving DNA double helix.

The Transmit DNA vibrations on the carrier, that resonates. The vibrations of the wearer become with the reflecting Laser beam recorded. The reflective laser beam arrives and reaches different photodetectors depending on the reflection angle. The data from the photodetectors are collected, stored and processed. This is done with the help of computers or computers and programs. The peaks are analyzed and the broken ones respectively A-T and G-C are assigned to the PER or APER sequence and thus become thus determining the resulting DNA sequence.

embodiment 2

Please refer 11a , The preparation and data analysis operations are similar to those described in Embodiment 1. The vibration is detected by atomic force microscopy.

embodiment 3

Please refer 10b , The preparation and data analysis operations are similar to those described in Embodiment 1. The detection of the vibrations takes place with the help of the field change analysis.

embodiment 4

Please refer 14a , The preparation and data analysis operations are similar to those described in Embodiment 1. The vibrations are detected by means of interference spectroscopy or interference analysis.

1

carrier

2

groove or depression

3

edge

4

dDNA or dsDNA

5

ssDNA

6

needle or partition

7

evaluation system

8th

top

9

electrical ladder

10

Non-wire

11

light source

12

photodetector

13

substratum

14

dsDNA fragment for ligation

15

optical fiber

16

Interference system

17

electrical management

18

laser beam

19

Laser source

20

Beam receiving and analysis system

21

electron beam

22

elastic Body, z. B. Resilinkörper

23

medium for DNA

24

denaturing Medium, such as. For example, ethanol

25

micropipette

26

magnet

27

immobilizer or immobilization site

28

Spiegelfeld

29

Gelrand

30

superconductors

31

boom or lever

32

carbon layer or carbon layer

33

airgel

34

Disk

35

Atomic Force Microscope

36

Surface, z. B. gold surface for measurement

37

string

38

Double Helix string

39

measuring body

40

Laser heating system or microwave generator

41

ice cream

42

medium for carriers, like Water, oil, lubricant

43

tub or container

44

electrical heating system

45

electrode

List of abbreviations

• ER-unwinding direction
• PER-parallel in / to the unwinding direction
• APER anti-parallel in / to the unwinding direction
• PERSeq-PER-sequence
• APERSeq-APER sequence
• PERSB-PER (sequence) specific area
• APERSB APER (sequence) specific area

Claims (13)

Method for sequence analysis of nucleic acids with the steps: a) isolation and spatial arrangement or orderly Laying or depositing a nucleic acid on a support, b) Unwinding of the nucleic acid, c) Determination of the unwinding vibrations of the nucleic acid - about the associated vibrations of the carrier means Laser beam reflection analysis, ion beam reflection analysis, electron beam reflection analysis, or and - by means of Field change analysis, Voltage variation analysis, Potential change analysis between the swinging carrier and a neighbor surface, or and - by means of Analysis of the current flow change through the carrier, or and - by electromagnetic detection methods, microscopy or atomic force microscopy, optical interference microscopy, scanning tunneling microscopy, optofaser interferometry, Spectral analysis, and / or - by Detection of mechanical oscillations, and / or - by thermosensory methods, as a single procedure or in Combination with each other, wherein the nucleic acid by vibration analysis is sequenced. The method of claim 1, wherein the entwined nucleic acid is preferred DNA, after step b) is returned in a controlled manner and orderly the nucleic acid with the method of claim 1c) is sequenced by vibration analysis. The method of claim 1, wherein as step b) a Unwinding and rewinding in the context of a replication of the nucleic acid, preferably DNA, expires and in this case the nucleic acid with the method of claim 1c) sequenced by vibration analysis becomes. Method according to one of the preceding claims, wherein the spatial Arrangement or the orderly laying or depositing of the nucleic acid spirally, wavy or straight on a support he follows. Method according to one of the preceding claims, wherein the deposition of the nucleic acid - by magnetic forces takes place, and wherein the magnet is located laterally or under the carrier, and / or - by electromagnetic forces takes place, and / or - by Adhesion forces or Cohesive forces of the aqueous medium the nucleic acid he follows. Method according to one of the preceding claims, wherein achieved the unwinding of the nucleic acid becomes a) by DNA helicases and ATP, or b) by a Replication machinery with DNA primases and DNA polymerases, or c) by adding denaturing substances, methanol, ethanol, mercaptoethanol, Alcohols, alkaline solutions, acid solutions, or d) by mechanical strand separation, or e) by Heating. Method according to one of the preceding claims, wherein the carrier is planar, or cone or columns wears and shape linear, angular, circular or disc-shaped is. Method according to one of the preceding Claims, wherein the carrier is connected by a resilient binding stud or lever with a solid substrate and is designed to vibrate. Method according to one of the preceding claims, wherein the carrier at least one groove for one nucleic acid contains. Method according to one of the preceding claims, wherein the carrier is applied to an elastic fabric or material, wherein the substance or material is on a substrate, and wherein the fabric or material is preferably made of resilin, elastin, Keratin, collagen, polyethylene. Method according to one of the preceding claims, wherein one end of the nucleic acid bound or immobilized or ligated to another nucleic acid is and thereby defines a certain unwinding direction is. Method according to one of the preceding claims, wherein the orderly laying or placing of the nucleic acid through a device is achieved using a pipette or micropipette connected with represents a magnet that can move or turn around to accelerate the spout at the top. device to carry out the method according to any one of the preceding claims, wherein at least one carrier for at least a nucleic acid is formed is, wherein a receiving or receiving device for vibrations of the carrier is formed, and wherein the reception of the vibrations on the Measuring method according to Claim 1c), individually or in combination with one another, based.