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T4 DNA ligase buffer solution for polymerase chain reaction in DNA coding compound library synthesis and application of T4 DNA ligase buffer solution

A compound library, enzyme chain reaction technology, applied in the field of biochemistry, can solve problems such as adverse effects of chemical reactions

Active Publication Date: 2019-03-08
上海药明康德新药开发有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The technical problem to be solved by the present invention is to find a suitable buffer formula by exploring the formula of the T4 DNA ligase buffer to overcome the defect that the residual components of the existing buffer enzyme chain reaction have an adverse effect on the next step of the chemical reaction

Method used

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  • T4 DNA ligase buffer solution for polymerase chain reaction in DNA coding compound library synthesis and application of T4 DNA ligase buffer solution
  • T4 DNA ligase buffer solution for polymerase chain reaction in DNA coding compound library synthesis and application of T4 DNA ligase buffer solution
  • T4 DNA ligase buffer solution for polymerase chain reaction in DNA coding compound library synthesis and application of T4 DNA ligase buffer solution

Examples

Experimental program
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Effect test

Embodiment 1

[0093] Example 1 10X T4 DNA Ligase Buffer Without DTT

[0094] 1. The ratio of 10X T4 DNA ligase buffer without DTT

[0095] 500mM Tris-HCl, 100mM MgCl 2 , 10 mM ATP (pH 8.0 at 25° C.).

[0096] 2. Preparation method of 10X T4 DNA ligase buffer without DTT

[0097] The buffer solution is entrusted to Nanjing Nuoweizan Biotechnology Co., Ltd. for exclusive use. The specific preparation method is as follows:

[0098] Accurately weigh 9.08g of trizma (Tris, CAS: 77-86-1) with an electronic balance, pour it into a clean 500mL beaker, and accurately measure 15mL of 1M MgCl with a 25mL pipette 2 , put it into the above beaker, accurately weigh 0.83g of adenosine triphosphate (ATP for short, CAS: 56-65-59000-83-3) with an electronic balance, add it to the above beaker, add to 120mL with Mill-Q water, Stir to dissolve, rinse the pH meter thoroughly with Milli-Q water and calibrate it to pass, add 5.7M HCl to adjust pH = 8.0 (25°C), use Milli-Q water to make up to 150mL, stir well ...

Embodiment 2

[0101] Example 2 Tris-free 2X T4 DNA ligase buffer

[0102] 1. Tris-free 2X T4 DNA ligase buffer ratio

[0103] 40mM HEPES, 20mM MgCl 2 , 2mM ATP, 8% 1,3-propanediol, 2mM DTT (pH 8.0at 25°C).

[0104] 2. Tris-free 10X T4 DNA ligase buffer preparation method

[0105] The buffer solution is entrusted to Nanjing Nuoweizan Biotechnology Co., Ltd. for exclusive use. The specific preparation method is as follows:

[0106] Accurately weigh 40mmol of HEPES with an electronic balance, pour it into a clean 1L beaker, and accurately measure 20mL of 1M MgCl with a 25mL pipette 2 , put into the above-mentioned beaker, accurately weigh 2mmol of adenosine triphosphate (adenosinetriphosphate, referred to as ATP, CAS: 56-65-59000-83-3), 80g of 1,3-propanediol, and 2mmol of DTT into the above-mentioned beaker with an electronic balance, Add to 800mL with Milli-Q water, stir to dissolve, rinse the pH meter thoroughly with Milli-Q water and calibrate it to pass, add 5M NaOH to adjust the pH =...

Embodiment 3

[0107] Example 3, Tris-free, DTT-free 2X T4 DNA Ligase Buffer

[0108] 1. Tris-free, DTT-free 2X T4 DNA ligase buffer ratio

[0109] 40mM EPPS, 20mM MgCl 2 , 2mM ATP, 8% 1,3-propanediol, 2mM DTT (pH 8.0at 25°C).

[0110] 2. Tris-free, DTT-free 2X T4 DNA ligase buffer preparation method

[0111] The buffer solution is entrusted to Nanjing Nuoweizan Biotechnology Co., Ltd. for exclusive use. The specific preparation method is as follows:

[0112] Accurately weigh 40mmol of EPPS with an electronic balance, pour it into a clean 1L beaker, and accurately measure 20mL of 1M MgCl with a 25mL pipette 2 , put it into the above beaker, accurately weigh 2mmol of adenosine triphosphate (ATP for short, CAS: 56-65-59000-83-3) with an electronic balance, add 80g of 1,3-propanediol into the above beaker, and use Mill-Q Add water to 800mL, stir to dissolve, rinse the pH meter thoroughly with Milli-Q water and calibrate it to pass, add 5M NaOH to adjust pH=8.0 (25°C), dilute to 1L with Mill...

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Abstract

The invention discloses three types of T4 DNA ligase buffer solutions for DNA coding compound library construction. The T4 DNA ligase buffer solutions include 1) DTT-free 10X T4 DNA ligase buffer solution in pH of 6.0-10.0 and prepared from 10-1000mM of Tris-HCl, 10-500mM of MgCl2 and 1-50mM of ATP, 2) Tris-free 2X T4 DNA ligase buffer solution in pH of 6.0-10.0 and prepared from 1-200mM of a sulfonic acid agent, 1-400mM of MgCl2, 0.1-5.0mM of ATP, 1-20% of 1,3-propanediol and 0.1-5.0mM of DTT and 3) Tris-free and DTT-free 2X T4 DNA ligase buffer solution in pH of 6.0-10.0 and prepared from 1-200mM of a sulfonic acid agent, 1-400mM of MgCl2, 0.1-5.0mM of ATP and 1-20% of 1,3-propanediol. The invention further discloses application of the three types of T4 DNA ligase buffer solutions. By the buffer solutions and application thereof, the defect of adverse effect, caused by residual components after existing buffer solution polymerase chain reaction, on next chemical reaction is overcome,and a DNA coding compound library synthesis period is shortened.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to the preparation and application of three T4 DNA ligase buffers used in the enzyme chain reaction in the synthesis of DNA coded compound libraries. Background technique [0002] Sydney Brenner and Professor Richard Lerner of the Scripps Research Institute in the United States proposed the concept of synthesis and screening of a DNA Encoded Library (DNA Encoded Library, DEL for short) in 1992 (references: Proc.Natl.Acad.Sci., 1992,89 , 5381, patent: US5573905), this method uses the "combination-separation" strategy of combinatorial chemistry by linking an organic small molecule reagent with a unique sequence of DNA at the molecular level (that is, DNA labeling the small molecule reagent) Quickly construct a huge number of compound libraries through two to more cycles, each compound in the compound library is composed of different organic small molecule reagent residues, and is identified ...

Claims

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Application Information

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IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 吴阿亮舒启胜安玉龙杨琪袁堂蜜龚平秀袁友浪张在红李科裴增飞陈雯婷蒯乐天杨洪芳彭宣嘉
Owner 上海药明康德新药开发有限公司
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