CN106191230A - The method that Deoxydization nucleotide carries out DNA sequencing is combined with aminoacid - Google Patents
The method that Deoxydization nucleotide carries out DNA sequencing is combined with aminoacid Download PDFInfo
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- CN106191230A CN106191230A CN201610084830.6A CN201610084830A CN106191230A CN 106191230 A CN106191230 A CN 106191230A CN 201610084830 A CN201610084830 A CN 201610084830A CN 106191230 A CN106191230 A CN 106191230A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/6869—Methods for sequencing
Abstract
The invention belongs to biological technical field, disclose a kind of DNA sequencing method of aminoacid Deoxydization nucleotide based on different electricity.The present invention utilizes aminoacid to build the deoxynucleoside acids thing of four kinds of different band electricity, four kinds of corresponding four kinds of different aminoacids of nucleotide, at least include an aminoacid, can also be the condensation polymer of the different length of multiple aminoacid composition, can be amino acid whose condensation polymer of the same race or the condensation polymer of different aminoacids；Then utilize Deoxydization nucleotide class Sihe DNA ligase that DNA profiling chain carries out order-checking to extend；The aminoacid that release is combined on dNTP during extending, utilizes amino acid whose different band electricity and combines the technology of semiconductor chips, being identified base, it is achieved determined dna sequence.This method breaches the limitation of the optical detection of DNA, it is possible to achieve the single-molecule sequencing of low cost, and method is simple and reliable.
Description
Technical field
The invention belongs to biological technical field, a kind of method relating to determined dna sequence, it is specifically related to a kind of nucleotide and combines the method that the aminoacid of different electricity carries out determining nucleic acid sequence.
Background technology
DNA (DNA (deoxyribonucleic acid)) is one of most basic material of composition organism, and the change of its sequence creates the natural world of nowadays this numerous and complicated beauty.Almost it may be said that the difference that all of biosis of the earth all derives from these bases of A, T, C, G puts in order, and the most insignificant random combine of this sequence, but contained quite abundant hereditary information and life intension.Just can effectively be obtained the nucleotide sequence of genome by sequencing technologies, greatly be convenient for people to recognize the diversity of life entity from gene level.Therefore, DNA sequencing is respectively provided with highly important meaning to life science, medical diagnosis on disease, personalized medicine etc..
Since the Human Genome Project completes, genomic sequencing technique has obtained swift and violent development.20 century 70 mid-terms, the first generation sequencing technologies that double deoxidating chain end cessation method is representative invented by Sanger helps people to complete from a large amount of examining orders such as phage genome to human genome sketch, but it is high to there is cost, speed is slow, the low deficiency that waits of flux, can not meet the needs of research application.Occur in that with the 454 of Roche after nineteen ninety-five, the Solexa of Solid and Illumina of the ABI secondary sequencing technologies as representative, it mainly forms complementary strand according to template sequence synthesis or hybridization, the fluorescently-labeled dNTP of different colours is introduced during complementary strand extends, sequence information is obtained by the different fluorescence signal of capture, and the PGM of Ion Torrent release in 2010, then utilize the proton that semiconductor chip detection base polyreaction produces, check order, feature is economical quick, but its accuracy is the highest.Checking order relative to a generation, secondary order-checking has had revolutionary change, and its flux, cost and speed are obtained for the lifting of brilliance, but its technical bottleneck is difficult to overcome, and especially template amplification and sequence is read long.Third generation order-checking with the SMRT technology of Pacific Bioscience as representative is also based on the principle of synthesis limit, limit order-checking, on polymerase active site, the dNTP of different colours fluorescent labeling phosphate group is combined one by one with template, produce fluorescence signal, read nucleic acid sequence information by obtaining fluorescence signal.Compared with secondary order-checking, third generation order-checking achieves single-molecule sequencing, has and need not amplification, reads the advantages such as long, but its accuracy rate need to improve.
List sequencing system at present, including the first generation, the second filial generation, the third generation, most is all fluorescent labeling dNTP using four kinds of different colours, identifies that different base, the namely acquisition of signal are realized by optical detection by dNTP from the fluorescence signal that matrix polymerization discharges during detection.And the technology such as the elimination that sensitivity and the comparison field of optics are disturbed are required the harshest by single molecule fluorescence detection, this is also the major reason that secondary order-checking cannot realize single-molecule sequencing before this.Although the RS system of Pacific Bioscience utilizes zero mould wave guide principles to eliminate background, but its instrument price is much more expensive, close to 1,000,000 dollars.
Being exactly price in addition with a factor the most crucial, the Human Genome Project carries out the stage in beginning, plans 3,000,000,000 dollars of gene order-checking work completing people, is equivalent to a base 1 dollar.Development along with sequencing technologies, nowadays the sequencing equipment of main flow completes a human needs 10,000 dollars, it can be seen that 6 orders of magnitude dropped in order-checking cost ratio before the more than ten years, but its large-scale application or limited, so have to realize breaking through, until perfect sequencing technologies occurs in existing technical system！
Summary of the invention
Gene sequencing can disclose the Deoxydization nucleotide of genomic DNA and put in order, and obtains biological heredity information.Biological and medical science tool are of great significance by the hereditary information obtaining biology fast and accurately.It is an object of the present invention to provide and a kind of combine the method that dNTP carries out DNA sequencing based on aminoacid, the present invention utilizes the aminoacid of different band electricity to distinguish different base, realize nucleotide sequence economy by the electric charge of detection amino acid group, measure rapidly, break the limitation of fluoroscopic examination, contribute to reducing order-checking cost.
DNA is made up of tetra-kinds of Deoxydization nucleotides of A, T, C, G, in order to distinguish these four Deoxydization nucleotide, needs to be marked so that it is be detected chip identification with different electric charges or different length marks.Different aminoacid is with the different quantities of electric charge, so the present invention designs aminoacid four deoxynucleotide phosphates of four kinds of band difference quantities of electric charge, wherein aminoacid is can leaving group, when DNA synthesizes, often close a Deoxydization nucleotide, the amino acid group that it is carried will leave away and enter the detection well on semiconductor chip, the amino acid group of different band electricity flows through the different changes of the electric current of field-effect transistor in causing well, thus captured by chip and identify, thus measure A, T, C, G and put in order.
For achieving the above object, technical solution of the present invention is: a kind of DNA sequencing method combining Deoxydization nucleotide based on aminoacid, comprises the following steps:
(1) dNTP is utilized to build aminoacid-deoxynucleotide phosphates (nucleotide analog), first phosphorylated amino acid is prepared, then the aminoacid of phosphorylation and the phosphate group of Deoxydization nucleotide are combined, at least include 2 phosphate groups, as shown in Figure 1.Wherein Base represents nucleoside base, and including A, T, G, C or U, AA represents the amino acid group of different band electricity, and n represents phosphate group number.-the OH of the aminoacid replacement nucleoside terminal phosphate group of different band electricity can obtain the nucleotide analog that at least four is amino acid modified；
Phosphate group number n of described different band electricity nucleotide analog can be 0,1,2,3,4...；
The amino acid groups such as described aminoacid can be lysine, arginine, histidine, tyrosine, cysteine, glutamic acid, aspartic acid；
Described aminoacid nucleotide analog at least includes an amino acid group, it is also possible to be multiple amino acids formed condensation polymers；
Described phosphate group and amino acid group can directly or indirectly combine；
Combine with lysine, arginine, glutamic acid, aspartic acid respectively as preferred A, T, C, G Deoxydization nucleotide and form deoxynucleotide analogs；
As preferably phosphoric acid group number >=2.
(2) moving in detection chip by the DNA profiling being combined with primer to be measured and buffer solution, DNA profiling to be measured enters in the well of chip, and each well accommodates zero or a DNA, well ad-hoc location fixed dna ligase, hatches base extension in 65 DEG C.
Described buffer solution comprises above-mentioned four kinds of aminoacid deoxynucleotide analogs, 20mM Tris-HCl, 50mM KCl, 5mM MgSO4, and 0.02%CA-630, pH 9.2；
Described DNA ligase is Therminator γ archaeal dna polymerase.
(3) principle based on the order-checking of synthesis limit, limit, when the complementary strand of DNA profiling often combines a upper base, key between this base 5 '-phosphoric acid and polyphosphoric acid is cut off, its amino acid group combined discharges, four kinds of bases discharge different amino acid groups respectively, the amino acid group discharged enters in the well 1 of semiconductor chip, the change of MOS memory 6 electric current under corresponding well is flow through by causing, by detecting the variable quantity of this electric current and recording this detection well location and put, determine amino acid group, and then may determine that corresponding base completes sequence.
Described aminoacid at least includes an amino acid group, four kinds of corresponding four kinds of different aminoacids groups of nucleotide, can also be multiple amino acid whose condensation polymers, can be amino acid whose condensation polymer of the same race or the condensation polymer of different aminoacids, four kinds of nucleotide can distinguish the amino acid condensation polymers of corresponding four kinds of different lengths.
Key alkali phosphatase between described δ phosphoric acid and amino cuts off, and discharges free amino acid group.
The quantity of electric charge that described different amino acid group is carried and the quantity of electric charge that the amino acid condensation polymers of different length is carried all differ.
Described semiconductor chip, including detection well 1, well sidewall SiO22, shaft bottom tantalum dioxide 3, MOS memory 5, and connect tantalum dioxide and one or more layers aluminum of MOS memory grid, copper or contain aluminum or the metal mixture 4 of copper.
(4) according to the curent change flowing through MOS memory caused by the quantity of electric charge of amino acid group, putting in order of base is obtained, it is thus achieved that DNA sequence information.
Combine, with aminoacid, the method that dNTP carries out DNA sequencing, in conjunction with the technology of semiconductor chips, complete gene sequencing by sensed current signal, there is no microscope and imaging device, simple in construction；Integrated level is higher, can the most multiple detections；And Single Molecule Detection can be realized, break through the bottleneck of existing optics order-checking, reduce rapidly order-checking cost.
Accompanying drawing explanation
Fig. 1 is amino acid tag Deoxydization nucleotide schematic diagram；Fig. 2 is semiconductor chip well schematic diagram；Fig. 3 is four kinds of monamino acid deoxynucleotide analogs: be respectively lysine four deoxyadenosine triphosphate, arginine four phosphoric acid deoxyribosylthymine, glutamic acid four deoxycytidine monophosphate, aspartic acid deoxyguanosine；Fig. 4 is lysine 8 aggressiveness, 12 aggressiveness, 16 aggressiveness and 20 aggressiveness four deoxynucleotide phosphates modified with coumarin: coumarin-8 polylysine-4 deoxyadenosine triphosphate, coumarin-1 2 polylysine-4 phosphoric acid deoxyribosylthymine, coumarin-1 6 polylysine-4 deoxycytidine monophosphate, coumarin-20 polylysine-4 guanosine 5-monophosphate.
Detailed description of the invention
In order to be better understood from the present invention, below in conjunction with the accompanying drawings, embodiment be further elucidated with the present invention, but present disclosure is not limited solely to example below.
Embodiment 1
In the present embodiment, the aminoacid deoxynucleotide analogs of four kinds of different band electricity of preparation, lysine four deoxyadenosine triphosphate (Lys-dA4P), arginine four phosphoric acid deoxyribosylthymine (Arg-dT4P), glutamic acid four deoxycytidine monophosphate (Glu-dC4P), aspartic acid four phosphate deoxidating deoxyguanosine (Asp-dG4P), as shown in Figure 3.First pass through aminoacid phosphorylation, then aminoacid and the nucleoside triphosphate of phosphorylation are combined and obtain required four kind aminoacid deoxynucleotide analogs.
Choose template strand 5 '-GCACCATATAATCGCTCGCATATTA-3 ', fix γ archaeal dna polymerase and polynucleotide template on a semiconductor die, add 1X Therminator gamma reaction buffer [50mM KCl, 20mM Tris-HCl, 5mM MgSO4,0.02%IGEPAL CA-630 (pH 9.2)].It is A that template initiates base, and its complementary base is T, therefore pipettes arginine four phosphoric acid deoxyribosylthymine as reaction substrate, then after fundamental chain in Arg-dT4P combination, cuts off N-P key release Arg with alkaline phosphatase, and the Arg of release enters the detection well of chip.The electric charge that amino acid group is carried, change is flow through the electric current of detection down-hole MOS memory, the curent change that the amino acid group of the band difference quantity of electric charge causes is different, detect this current change quantity and record well location and put and just amino acid group kind can be identified, outgoing signal, it is thus achieved that base information.
Embodiment 2
In the present embodiment, be combined formation aminoacid deoxynucleotide analogs using the condensation polymer of lysine with dNTP as mark.Lysine 8 aggressiveness, 12 aggressiveness, 16 aggressiveness and 20 aggressiveness that selection is modified with coumarin are combined with four deoxynucleotide phosphates modified with diaminoheptane, form coumarin-8 polylysine-4 deoxyadenosine triphosphate, coumarin-1 2 polylysine-4 phosphoric acid deoxyribosylthymine, coumarin-1 6 polylysine-4 deoxycytidine monophosphate, coumarin-20 polylysine-4 guanosine 5-monophosphate respectively, as shown in Figure 4.
Remaining step of the present embodiment is similar to Example 1, and remaining difference is for replace with multiple aminoacid by single amino acids, and the effect that can realize signal amplification during detection and the warm-up movement speed weakening molecule are easy to detection.
The possible embodiments that the foregoing is only the present invention illustrates, and this embodiment is also not used to limit the scope of the claims of the present invention, all impartial changes made according to scope of the present invention patent and modification, all should belong to the covering scope of patent of the present invention.
Claims (10)
1. a DNA sequencing method for aminoacid Deoxydization nucleotides based on different electricity, the aminoacid deoxynucleotide analogs can left away including structure,
AA-dNTP:A, T, C, G or U are respectively in connection with the amino acid group of different band electricity；When carrying out template extension, base is attached to template
On discharge its entrained amino acid group；Then reach to distinguish the purpose of base by detection amino acid group, complete DNA sequencing.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that amino acidic group
Group is as the mark of Deoxydization nucleotide, it is also possible to is other charged groups, or has other different qualities, such as different size or difformity
Amino acid group.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that phosphate group
Directly or indirectly be combined with amino acid group, by cleavage, the marks such as aminoacid left away from order-checking synthesis chain.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that phosphate group
Number >=2.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that phosphate group
Can be joined directly together with amino acid group, it is also possible to be connected by linking group, such as double amino alkanes etc..
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that at least include
One aminoacid, it is also possible to be multiple amino acid whose condensation polymers, or the mixing condensation polymer that multiple different aminoacids is formed.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that described DNA
Ligase is Therminator γ archaeal dna polymerase.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, it is characterised in that mark is
Charged aminoacid or other charged groups, can obtain mark kind and the material of signal intensity by electrical signal detection.
The DNA sequencing method of a kind of aminoacid Deoxydization nucleotides based on different electricity the most according to claim 1, after amino acid group is released
Entering the detection well of IC chip, the amino acid group of different band electricity causes the change of detection down-hole MOS memory electric current
Change, by detection current change quantity and detection well position in IC chip, reach to distinguish the purpose of base, complete DNA sequencing.
Semiconductor chip the most according to claim 9, it is characterised in that detection shaft bottom material is the compounds such as tantalum dioxide, connect tantalum dioxide and
MOS memory grid is one or more layers aluminum, copper or containing aluminum or the metal mixture of copper；Detection chip at least includes a well
Array of structures.
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| CN1526829A (en) * | 2003-03-04 | 2004-09-08 | 中国人民解放军基因工程研究所 | Quick detection method of pathogenic microbe diagnosis type gene chip |
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