阅读说明：本技术 一种核酸探针以及一种核酸测序方法 (Nucleic acid probe and nucleic acid sequencing method ) 是由 刘二凯 章文蔚 陈奥 徐崇钧 于 2018-07-06 设计创作，主要内容包括：核酸探针以及边连接边测序的核酸测序方法,上述核酸探针为DNA测序探针,其包含第一部分、第二部分、连接体和可检测的标记物,第一部分的碱基为A、T、U、C或G,第二部分的碱基为随机碱基和/或通用碱基,并且至少为3个碱基,第一部分和第二部分通过连接体连接,并且第一部分和连接体之间的连接能够被切断,可检测的标记物连接在第二部分或者连接体上。上述探针或将其组合或者测序方法能够降低测序中的探针数量或种类,从而降低成本。(A nucleic acid probe which is a DNA sequencing probe comprising a first portion, a second portion, a linker and a detectable label, wherein the first portion has A, T, U, C or G bases, the second portion has at least 3 bases which are random bases and/or universal bases, the first portion and the second portion are linked by the linker, the linkage between the first portion and the linker can be cleaved, and the detectable label is linked to the second portion or the linker. The probes or the combination thereof or the sequencing method can reduce the number or the types of the probes in sequencing, thereby reducing the cost.)

A nucleic acid probe comprising a first portion, a second portion, a linker, and a detectable label, wherein:

the first part has bases A, T, U, C or G,

the bases of the second portion are random bases and/or universal bases and are at least 3 bases,

the first part and the second part being connected by a connecting body, and the connection between the first part and the connecting body being capable of being severed,

the detectable label is attached to the second moiety or linker.

The nucleic acid probe of claim 1, wherein the first moiety is located at the 5 'end or the 3' end.

The nucleic acid probe according to claim 1, wherein the bases of the second portion are 3 to 15 bases, preferably 5 to 12 bases, more preferably 5 to 10 bases (e.g. 5, 6, 7, 8, 9 or 10 bases), and particularly preferably 6 to 9 bases.

The nucleic acid probe of claim 1, wherein the detectable label is a fluorophore; preferably at least one selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF 688;

preferably, the detectable label is attached to the second moiety;

preferably, the detectable label is attached to the 3' -OH at the end of the second moiety;

preferably, the detectable label is linked to the 3' -OH at the end of the second moiety by a phosphoester bond.

The nucleic acid probe according to claim 1, wherein the linker does not contain a sulfur atom; preferably, the linker is selected from the group consisting of formula IV-formula IX below:

wherein, in the formula IV, R¹Selected from H, OH, C₁-C₆Alkyl radical, C₂-C₆Alkenyl and C₂-C₆An alkynyl group; r²Selected from H, OH, F, Cl and Br.

A nucleic acid probe combination comprising 4 sets of probes, wherein:

a first set of nucleic acid probes: comprising the nucleic acid probe of any one of claims 1 to 5, wherein the base of the first portion is A;

a second set of nucleic acid probes: comprising the nucleic acid probe of any one of claims 1 to 5, wherein the base of the first portion is T or U;

third set of nucleic acid probes: comprising the nucleic acid probe of any one of claims 1 to 5, wherein the base of the first portion is C;

fourth set of nucleic acid probes: comprising the nucleic acid probe of any one of claims 1 to 5, wherein the base of the first portion is G;

and the detectable labels in the 4 sets of nucleic acid probes are different from each other;

the 4 sets of nucleic acid probes are mixed or not mixed;

preferably, the number of moles of the first set of nucleic acid probes is equal to the number of moles of the fourth set of nucleic acid probes;

preferably, the number of moles of the second set of nucleic acid probes is equal to the number of moles of the third set of nucleic acid probes;

preferably, the sum of the number of moles of the first set of nucleic acid probes and the number of moles of the fourth set of nucleic acid probes is less than or equal to the sum of the number of moles of the second set of nucleic acid probes and the number of moles of the third set of nucleic acid probes;

preferably, the first set of nucleic acid probes: a second set of nucleic acid probes: third set of nucleic acid probes: the molar ratio of the fourth group of nucleic acid probes is (0.5-2): (3-5): (3-5): (0.5-2); more preferably 1: 4: 4: 1.

a ligation solution comprising the nucleic acid probe of any one of claims 1 to 5 or the combination of nucleic acid probes of claim 6, and a DNA ligase.

The connecting fluid according to claim 7, characterized by any one or more of the following (1) to (5):

(1) the DNA ligase is one or more selected from T4DNA ligase, T7DNA ligase and T3DNA ligase;

(2) the concentration of the nucleic acid probe is 0.1-5 mu M, preferably 1 mu M;

(3) the concentration of the DNA ligase is 0.01-2 mu M, preferably 0.5 mu M;

(4) also comprises the following components:

50mM CH₃COOK，20mM Tris，10mM Mg(CH₃COO)₂，100μg/ml BSA，1mM ATP，10％PEG6000；

(5) the balance of the connecting liquid is water.

A kit comprising the nucleic acid probe of any one of claims 1 to 5, or the combination of nucleic acid probes of claim 6, or the ligation solution of claim 7 or 8;

preferably, further comprising one or more selected from the group consisting of a reagent capable of cleaving the linkage between the first moiety and the linker, a buffer for solubilizing the nucleic acid probe, and a sequencing primer;

preferably, the reagents in the kit do not contain silver ions.

The kit of claim 9, wherein the reagent capable of cleaving the linkage between the first moiety and the linkerIs endonuclease (such as endonuclease IV or endonuclease V), organic phosphide (such as THPP or TCEP) or PdCl₂And sulfonated triphenylphosphine complexes.

A method of nucleic acid sequencing comprising the steps of:

(1) hybridizing a sequencing primer to a nucleic acid molecule to be detected;

(2) ligating the nucleic acid probe of any one of claims 1 to 5 or the combination of nucleic acid probes of claim 6 to a sequencing primer;

(3) eluting the nucleic acid probes which are not combined with the nucleic acid molecules to be detected;

(4) detecting the detectable label on the nucleic acid probe bound to the nucleic acid molecule to be detected, and determining the base information of the first portion;

(5) cleaving the first portion of the nucleic acid probe from the linker and eluting the remaining portion of the nucleic acid probe excluding the first portion;

preferably, the method further comprises the following steps:

(6) repeating the aforementioned steps (2) - (4) or (2) - (5).

The sequencing method according to claim 11, wherein in step (2), the nucleic acid probe according to any one of claims 1 to 5 or the nucleic acid probe combination according to claim 6 is ligated to a sequencing primer by using the ligation solution according to claim 7 or 8.