Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing

A fluorescent labeling and terminal technology, applied in the fields of chemical synthesis and biochemistry, can solve technical difficulties and other problems

Active Publication Date: 2014-01-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although single-molecule sequencing technology has commercialized products, there are still technical difficulties and cannot be applied on a large scale
[0004] At present, the high-throughput sequencing platform on the market is monopolized by a few foreign products. What is particularly

Method used

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  • Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing
  • Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing
  • Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

[0131]

[0132] The corresponding synthetic route is as figure 2 Shown; specifically include the following steps:

[0133] 1.1 Compound F 2 Synthesis

[0134] Methyl trifluoroacetate reacts with propargylamine in an organic solvent to obtain compound F 2 , specifically: add 60ml of methanol to a single-necked bottle, stir under an ice-water bath, add propargylamine (60mmol, 3.3042g), stir for 15 minutes and then slowly add methyl trifluoroacetate (86.7mmol, 11.0957g) for 10 minutes Afterwards, the ice-water bath was removed, and the reaction was carried out at room temperature for 24 hours. The reaction was monitored with a TLC plate, PE: EA = 8: 1, baking plate, Rf = 0.5, a new spot was generated as product F2. Distillation under reduced pressure (51°C, 280Pa) yielded 3.53g with a yield of 39%.

[0135] 1 H NMR (CDCl 3 , 300MHz): δ2.32(t, J=4.0Hz, ...

Embodiment 2

[0161] The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

[0162]

[0163] The corresponding synthetic route is as Figure 5 Shown; specifically include the following steps:

[0164] 2.1 Compound F 2 , F 3 The synthesis is the same as in Example 1

[0165] 2.2 Synthesis of compound G1

[0166] Take 23mg F 3 (0.06mmol) in a 10mL single-necked bottle, add 1mL of methanol to dissolve, add 0.1mL of concentrated ammonia (6mmol), and stir overnight at room temperature. TLC plate monitoring: DCM:MeOH=3:1, product G1Rf=0.15. Separation adopts TLC plate chromatography, MeOH:EA:NH3=6:6:1, collect Rf=0.6 ultraviolet color region. ESI-HRMS: cals for C 12 h 15 N 3 o 5 [M] 281.1012, found 281.1015.

[0167] In the above synthesis, the ammonia water added can be any value in 3-6 mmol.

[0168] 2.3 Synthesis of compound G2

[0169] Take 8.5mg G1 (0.03mmol) and dissolve it with 0.5mL methanol; take 9.4mg SPDP (0.03mmol...

Embodiment 3

[0181] The structural formula of the reversible terminal of this embodiment is shown in formula (III):

[0182]

[0183] The corresponding synthetic route is as Figure 7 As shown, the specific steps are as follows:

[0184] 3.1 Synthesis of Compound N-1

[0185] Take a 100ml one-mouth bottle, add 0.75g (6.6mmol) of cysteamine hydrochloride, dissolve it with 4ml of methanol, and add 2.04g (6.6mmol, 50% aqueous solution, Dissolve in 3ml of methanol) and 1.85ml of TEA (13.2mmol) mixture, remove the ice-water bath after 30min, and stir at room temperature. TLC tracked the reaction process, stopped the reaction after 24h, spin out the solvent, and plate chromatography, MeOH:EA=1:1, 44mg of the product was obtained as a yellow oily liquid.

[0186] 1 H NMR (D 2 O, 400MHz): δ2.92(t, J=6.0Hz, 2H), 3.00(t, J=6.4Hz, 2H), 3.40(t, J=6.4Hz, 2H), 3.87(t, J=6.0 Hz, 2H).

[0187] In the above synthesis, the added 2-hydroxyethyl disulfide can be any value in 6.6-13.2 mmol, and the T...

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Abstract

The invention discloses a four-color fluorescence labeling reversible terminal and a use thereof in DNA (Deoxyribonucleic Acid) sequencing. The structural formula of the reversible terminal is as shown in a formula (I) in the specification, wherein R1 is triphosphate; R2 is H or OH; the basic group is U, C, A, G or derivatives thereof; the connecting unit is a bifunctional compound which is breakable under a mild condition; the fluorescence group is one selected from a combination of BODIPY, fluorescein, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, alexa, squarene dye and an energy transferring dye, and derivatives thereof. The reversible terminal provided by the invention can be used for DNA single-molecule sequencing; simultaneously, raw materials required by the synthesis of the reversible terminal provided by the invention are simple and easy to get and the synthesis process of the reversible terminal is completely involved with conventional chemical reactions, so that the four-color fluorescence labeling reversible terminal can be popularized and utilized to a large scale; biological evaluation results indicate that the reversible terminal is capable of completely meeting the biochemical reaction requirements of high-flux sequencing and has a good practical prospect.

Description

technical field [0001] The invention relates to the fields of chemical synthesis and biochemistry, in particular to a kind of four-color fluorescent label reversible terminal and its application in DNA sequencing. Background technique [0002] DNA sequencing technology is one of the important means of modern life science and medical research. DNA sequencing started with Sanger sequencing technology (generation sequencing) in 1977, and has developed rapidly in the past thirty years. The throughput of sequencing has been greatly increased and the cost has dropped sharply. Some people even think that the speed of its development has broken the existing Moore's Law budget in the semiconductor industry. The emergence of the second-generation high-throughput parallel sequencing technology is a concentrated expression of the rapid development of sequencing technology. Using first-generation sequencing technology, the Human Genome Project (HGP) spent $3 billion to sequence the ent...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07H19/10C07H19/14C07H1/00C12Q1/68
Inventor 沈玉梅邵志峰赵小东汤道年龚兵李小卫江敏刘亚智魏晓飞伍新燕
Owner SHANGHAI JIAO TONG UNIV
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