GB/T 39512-2020.Principle for phosphorylation labeled nucleic acid testing.
1Scope
GB/T 39512 specifies the terms and definitions, abbreviations, general requirements, determination methods, sample storage and reporting for phosphorylation labeled nucleic acid testing.
GB/T 39512 is applicable to the detection of phosphorylation labeled nucleic acids for the construction of DNA-encoded compound libraries.
2Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water used in analytical laboratories
GB/T 30988 Extraction, purification and test methods for genomic DNA of polyphenolic plants
GB/T 30989 Technical specifications for high-throughput gene sequencing
GB/T 34797 Technical requirements for quality of nucleic acid primers and probes
3 Terms and definitions
The terms and definitions defined in GB/T 34797 and the following terms and definitions apply to this document.
3.1
Phosphorylation labeled nucleic acid
Single-stranded DNA with a phosphate group labeled at the 5' end and a length ranging from 9 nt to 17 nt.
3.2
DNA encoded compoundDNA encoded compound
Having a phosphate group labeled at the 5' end and a 2-6 base sticky end at the 3' end, two single-stranded DNAs of 9 nt to 17 nt are annealed to form a double strand.
3.3
Annealing
Two single-stranded DNAs are bound together by heating for denaturation and cooling for renaturation according to the principle of complementary base pairing.
3.4
?? Complementary strand
Two single strands in a double-stranded nucleotide chain formed by complementary base pairing.
4 Abbreviations
The following abbreviations apply to this document.
DIEA: N,N-diisopropylethylamine
DNA: Deoxyribonucleic Acid
EDTA: Ethylenediaminetetraacetic Acid
This standard specifies the terms and definitions, abbreviations, general requirements, determination methods, sample storage and reporting for the detection of phosphorylated labeled nucleic acids. This standard applies to the detection of phosphorylated labeled nucleic acids used in the construction of DNA-encoded compound libraries.

ICS07.080
National Standard of the People's Republic of China
GB/T39512—2020
Principle for phosphorylation labeled nucleic acid testing2020-11-19Issued
State Administration for Market Regulation
National Standardization Administration
Issued
2021-06-01Implementation
Foreword
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). GB/T39512—2020
The drafting units of this standard are: Biological Research Institute of China Testing Technology Research Institute, General Biosystems (Anhui) Co., Ltd., Sichuan Yazhong Gene Technology Co., Ltd., Shenzhen Metrology and Quality Inspection Institute, China Testing Technology Research Institute, Chengdu Pioneer Pharmaceutical Development Co., Ltd., Suzhou Jinweizhi Biotechnology Co., Ltd., West China Hospital of Sichuan University, Sichuan Agricultural University, Hebei Medical University. The main drafters of this standard are: Zhou Lihua, Yong Jingui, Han Guoquan, Liu Zongwen, Yang Jiebin, Pan Hong, Li Huaiping, Zhang Yi, Ma Lixia, Yang Guowu, Liu Qian, Jiang Zijing, Yang Li, Hao Junzheng, Zheng Yanyan, and Lv Pin. 1
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1Scope
General rules for phosphorylation-labeled nucleic acid detection
GB/T39512—2020
This standard specifies the terms and definitions, abbreviations, general requirements, determination methods, sample storage and reporting for phosphorylation-labeled nucleic acid detection. This standard applies to the detection of phosphorylated labeled nucleic acids used in the construction of DNA-encoded compound libraries. Normative references
The following documents are essential for the application of this document. For dated references, only the dated version applies to this document. For undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water for analytical laboratories GB/T30988
GB/T30989
GB/T34797
Terms and definitions
Extraction, purification and test methods of genomic DNA from polyphenolic plants High-throughput gene sequencing technology procedures
Quality technical requirements for nucleic acid primers and probes
The terms and definitions defined in GB/T34797 and the following terms apply to this document. 3.1
phosphorylation labeled nucleic acid
Single-stranded DNA with a phosphate group labeled at the 5' end and a length ranging from 9nt to 17nt. 3.2
DNAencodedcompound
DNAencodedcompound
Having a phosphate group labeled at the 5° end, 2 to 6 base sticky ends at the 3 end, and a double strand formed by annealing two single-stranded DNAs of 9nt to 17nt.
Annealing
Two single-stranded DNAs are combined together according to the principle of complementary base pairing by means of heating denaturation and cooling renaturation. 3.4
complementarystrand
complementarystrand
Two single strands in a double-stranded nucleotide chain formed by complementary base pairing. Abbreviations
The following abbreviations apply to this document.
DIEA: N, N-diisopropylethylamine (Disopropylethylamine) DNA: Deoxyribonucleic Acid (Deoxyribonucleic Acid) EDTA: Ethylenediaminetetraacetic Acid (Ethylenediaminetetraacetic Acid) rKaeerKAca-
GB/T39512—2020
HFIPA: Hexafluoroisopropanol (Hexafluoroisopropanol) HPLC High Performance Liquid Chromatography (High Performance Liquid Chromatography) LC-MS: Liquid Chromatography-Mass Spectrometer (Liquid Chromatography-Mass Spectrometer) OD: Optical Density (Optical Density) TEAA: Triethylammonium Acetate (Triethylammonium Acetate) 5 Phosphorylation Labeling Nucleic Acid Detection General Requirements
Sample Requirements
The test sample should be a colorless or light yellow clear liquid, without suspended matter and mechanical impurities. The sample tube should be well preserved without damage or leakage. Personnel requirements
Personnel conducting phosphorylated labeled nucleic acid detection should have an educational background with professional knowledge in biology and chemistry. Reagent requirements
Unless otherwise specified, the reagents used in the test are analytically pure reagents, and the water is the first-class water specified in GB/T6682. 5.4 Test items
For the test items of phosphorylated labeled nucleic acids, please refer to Table 1, and for the reference results of the index parameters, please refer to Appendix A. Table 1 Phosphorylated labeled nucleic acid detection indicators and measurement instrumentsSample
Phosphorylated labeled nucleic acid
Purity
Indicator items
Total amount
Relative molecular mass
Base accuracy
Ultraviolet absorption intensity
HPLC purity
Mass spectrometry purity
Base deletion rate
Complementary chain concentration difference
Dephosphorylated products||t t||Joining efficiency
Complete match
Base deletion rate
UV absorption intensity
Mass spectrum peak intensity ratio
Concentration difference between compounds
Dephosphorylation product ratio
Raw material gel image conversion rate/
Relative molecular weight
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Determination method
6.3.2
6.3.3
6.4.1
6.4.2
6.4.3
6.5.2
6.5.1
6.6.1
6.6.2
6.6.3
Instruments
UV spectrophotometer
LC-MS
LC-MS
Sequencer
UV spectrophotometer Analyzer
HPLC instrument
LC-MS
Sequencer
LC-MS
Ultraviolet spectrophotometer
LC-MS
Ultraviolet spectrophotometer
LC-MS
Gel electrophoresis instrument
LC-MS
6 Determination method
Total amount detection
Carry out in accordance with GB/T34797.
Relative molecular mass detection
6.2.1 Direct calculation
Relative molecular mass can be directly calculated according to the customized sequence according to formula (1): GB/T39512-2020
MW=(AX313.21)+(CX289.18)+(GX329.21)+(TX304.2)+79.98-61.94(1)
Wherein:
Relative molecular mass;
The number of bases corresponding to each base of A, C, G, T; 79.98
61.94
5Relative molecular mass of phosphate group;
Relative molecular mass of phosphodiester bond residue. 6.2.2 Mass spectrometry detection
LC-MS is used to detect the relative molecular mass (MW) of phosphorylated labeled nucleic acid. LC-MS converts the sample into moving charged ion fragments through the electrospray ionization source, and then separates and records them according to the mass-to-charge ratio (m/), and the relative molecular mass of the target compound is calculated by mass spectrometry analysis software. Before the instrument is operated, check and confirm that the instrument's ion source, detector, data system, etc. are in normal condition. The equipment parameters and mobile phase parameters of LC-MS refer to B.1 in Appendix B. 6.2.3 Determination results
The relative molecular mass (MW) measured by method 6.2.2 is compared with the relative molecular mass (MW) calculated in 6.2.1 to obtain the relative error.
6.3 Base accuracy detection
6.3.1 General
The degree of matching between the phosphorylated labeled nucleic acid sequence and the customized sequence·The sequence information automatically generated by the synthesizer is compared and verified with the results of mass spectrometry detection and sequencing detection.
6.3.2 Liquid chromatography-mass spectrometry
Use the method in 6.2.2 to determine, and compare and verify the measured relative molecular mass with the theoretical relative molecular mass (MW) of the customized sequence in 6.2.1.
6.3.3 Gene sequencing
Perform in accordance with GB/T30989, and perform sequence determination on phosphorylated labeled nucleic acids. 6.4 Purity detection
6.4.1 Ultraviolet spectrophotometry
Perform in accordance with GB/T30988.
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GB/T39512—2020
6.4.2 High performance liquid chromatography
Use HPLC to determine the purity of phosphorylated labeled nucleic acids, and refer to B.2 for equipment operating parameters. After ensuring that the instrument is in normal condition, take 10μL of 50μmol/L phosphorylated labeled nucleic acid solution and 20uL of ultrapure water and mix them in the injection bottle. Click the instrument run button and the instrument will automatically inject and test. According to the test results, calculate the ratio of the target peak area to the total peak area to obtain the purity of the phosphorylated labeled nucleic acid. 6.4.3 Liquid chromatography-mass spectrometry
Use the method in 6.2.2 to determine, calculate the ratio of the intensity of the mass spectrometry signal of the target peak to the intensity of the mass spectrometry signal of all peaks, and obtain the mass spectrometry purity of the phosphorylated labeled nucleic acid.
6.5 Base deletion rate detection
6.5.1 Liquid chromatography-mass spectrometry
Use the method in 6.2.2 to determine, calculate the ratio of the mass spectrometry signal intensity of the base-deficient peak to the mass spectrometry signal intensity of all peaks, and obtain the base deletion rate.
6.5.2 Gene sequencing method
Use the method in 6.3.3 to determine and calculate the base deletion signal ratio. 6.6 Complementary chain concentration difference detection
6.6.1 UV absorption intensity detection
6.6.1.1 Single-chain concentration detection
Use UV spectrophotometry to detect the UV absorption intensity of phosphorylated labeled nucleic acid, and measure the absorption intensity of nucleic acid at 260nm, i.e. OD260 value. The measured OD26 value multiplied by the dilution factor is used as the final quantitative OD260 value, and the OD260 value is divided by the extinction coefficient to convert it into sample concentration.
6.6.1.2 Complementary chain concentration difference
Calculate the concentration difference between the two complementary chains based on the single-chain quantitative results. Note: Centrifuge and mix the samples before detection. When the sample volume is large, use a shaker at 600.r/min and 25℃ for 10 min to mix. 6.6.2 Mass spectrometry peak intensity ratio detection
Use the method in 6.2.2 to determine and calculate the ratio of the mass spectrometry signal intensity of the single strand with lower content in the double strand to the total mass spectrometry signal intensity of the double strand. 6.6.3 Detection of concentration difference between compounds
Use ultraviolet spectrophotometry to determine the ultraviolet absorption intensity of the phosphorylated labeled nucleic acid double strands formed after annealing of the complementary strands. Divide the detected mass concentration by the relative molecular mass of the complementary strand mixed product to obtain the molar concentration. Finally, calculate the molar concentration difference between different phosphorylated labeled nucleic acid double strands, that is, the concentration difference between compounds. 6.7 Detection of dephosphorylated products
Use the method in 6.2.2 to determine and calculate the ratio of the mass spectrometry signal intensity of the product peak without labeled phosphate group (whose relative molecular mass is 79.98 lower than the target relative molecular mass) to the signal intensity of all mass spectrometry peaks to obtain the dephosphorylation rate. 4
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6.8 Ligation efficiency detection
6.8.1 Sample preparation
GB/T39512—2020
The ligation efficiency was determined by gel electrophoresis. 6.5μL1mmol/L quality inspection substrate, 2μL10X ligation buffer and 2.5μgT4DNA ligase were added to 5uL1mmol/L phosphorylated labeled nucleic acid stock solution, and the volume was fixed to 20μL and mixed to prepare the reaction solution. The prepared reaction solution was placed at 20℃ for 4h. After the reaction, 2μL of the reaction solution was diluted 25 times and set aside. 6.8.2 Ligation efficiency detection
The reaction solution was subjected to agarose gel electrophoresis; the electrophoresis results were used to calculate the ligation efficiency using the grayscale quantitative method, where the ligation efficiency = grayscale value of the ligation product/(grayscale value of the ligation product minus grayscale value of the remaining raw material). Sample preservation
Store in a -20℃ refrigerator to avoid repeated freezing and thawing. Undiluted dry powder should not be stored at -20℃ for more than 1 year; diluted samples should not be stored at -20℃ for more than half a year.
Report
After the synthesis of phosphorylated labeled nucleic acid, a synthesis report or equivalent guidance document should be attached. The content of the document should at least include the following parts: name;
a)
b) sequence;
length;
purification method;
molar weight;
relative molecular weight;
concentration;
g)
storage conditions and validity period.
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GB/T39512—2020
Appendix A
(Informative Appendix)
Indicators and reference results for detection of phosphorylated labeled nucleic acids For the indicators and reference results of detection of phosphorylated labeled nucleic acids, please refer to Table A.1. Table A.1 for indicators and reference results for detection of phosphorylated labeled nucleic acids
Sample
Phosphorylated labeled nucleic acid
Purity
Base deletion rate
Concentration difference of complementary chains
Dephosphorylated products
Joining efficiency
Indicators
Total amount
Relative molecular weight
Base accuracy
Ultraviolet absorption intensity
Chromatographic purity
Mass spectrometry purity
Complete match
Base Base missing rate
UV absorption intensity
Mass spectrum peak intensity ratio
Concentration difference between compounds
Dephosphorylation product ratio
Raw material gel map conversion rate/
Relative molecular weight
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Reference result
Deviation ≤±15%
Deviation ≤0.05%
Deviation ≤0.05%
Consistent with customized sequence
OD260/OD280:1.71.9||tt ||OD260/OD230>1.7
>85%
Deviation<±15%
>20%
Deviation≤25%
>90%
Liquid chromatography-mass spectrometry
Appendix B
(Informative)
Equipment and conditions for LC-MS and HPLCbzxZ.net
The mobile phase preparation and detection conditions for liquid chromatography-mass spectrometry are as follows: a)
The mobile phase preparation for liquid chromatography-mass spectrometry refers to Table B.1: Mass spectrometry conditions: electrospray ionization source, ion trap mass analyzer; capillary voltage: 4000 V;
Nebulizer gas pressure: 0.28 MPa;
Nebulizer gas temperature: 350°C;
Burst voltage: 150 V;
Nebulizer gas: ammonia;
Chromatographic column: CisColumn, 130A, 2.5μm, 4.6mmX50mm; Column temperature: 40°C;
Flow rate: 0.5mL/min;
For gradient elution conditions, see Table B.2.
Table B.1
Preparation of mobile phase A and mobile phase B
Mobile phase A
(calculated in IL)
Mobile phase B
(calculated in 1L)
0.075%HFIPA
(750μL)
0.075%HFIPA
(750μL)
0.0375%DIEA
(375μL)
0.0375%DIEA
(375μL)
Table B.2
Time
High performance liquid chromatography
The detection conditions of high performance liquid chromatography are as follows: 10μmol/L EDTA
10mL 1mmol/L EDTA
10 μmol/L EDTA
10mL1mmol/EDTA
Gradient elution conditions (LC-MS)Mobile phase A volume fraction
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GB/T39512—2020
100%H.0
(990mLH,O)
80%ACN.20%H.0
(800mLACN.190mLH,0)
Mobile phase B volume fraction
GB/T39512-—2020
Chromatographic column: CigColumn.130A, 2.5μm, 4.6mmX50mm; mobile phase A: 0.1mol/LTEAA; mobile phase B: acetonitrile; column temperature: 60℃;
detector: UV detector;
detection wavelength: 260nm;
flow rate: 1.0mL/min;
gradient elution conditions refer to Table B.3.
Table B.3
time
10.5
11.5
gradient elution conditions (high performance liquid chromatography) mobile phase A volume fraction
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mobile phase B volume fraction
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