Primer probe composition for detecting cell genetic stability and application thereof

A technology of genetic stability and primer probe, which is applied in the field of primer probe composition to detect the genetic stability of recombinant CHO cells, can solve problems such as the influence of quantitative results of target genes, and achieve low cost, high accuracy and strong specificity Effect

Pending Publication Date: 2021-08-06
TOT BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purpose of this method is to detect the residual DNA content of CHO cells, and in the process of quantifying the target gene, an internal reference gene is added to achieve relative quantification of the target gene. The quantitative results of the

Method used

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  • Primer probe composition for detecting cell genetic stability and application thereof
  • Primer probe composition for detecting cell genetic stability and application thereof
  • Primer probe composition for detecting cell genetic stability and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Preparation of VEGF light chain gene standard

[0058] (1) Preparation of light chain plasmid DNA

[0059] According to the VEGF light chain gene design primer pair shown in SEQ ID NO:1~2 and the probe shown in SEQ ID NO:3;

[0060] Use Invitrogen’s Purelink Genomic DNA kits to extract CHO cell genomic DNA, use the primer pair VEGFLF (SEQ ID NO: 1) and VEGFLR (SEQ ID NO: 2) to amplify the complete VEGF light chain gene, and the PCR reaction system is: 2× PrimeSTAR Max Premix, 25 μL; VEGFLF (10 nM), 1 μL; VEGFLR (10 nM), 1 μL; ddH 2 O, 21 μL; genomic DNA, 2 μL; reaction conditions: 98°C for 10 min; 98°C for 1 min, 57°C for 5 s, 72°C for 90 s, a total of 30 cycles;

[0061] Use Taq enzyme to add poly A tail reaction to the amplified product. The reaction system is: Taq enzyme, 0.2 μL; 10×Taq enzyme Buffer, 1 μL; dNTP (10 mM), 0.5 μL; PCR product, 8.3 μL; the reaction conditions are: 72°C, 10-15 minutes;

[0062] The poly A-tailed PCR product was TA-ligated w...

Embodiment 2

[0069] Example 2 Establishment of the amplification standard curve of VEGF light chain gene

[0070] According to the plasmid copy number formula in step (2) of Example 1, the copy number of 1 μ L of light chain plasmid standard was calculated to be 1.95 × 10 11 , according to the amplification efficiency and CT value, the initial concentration of the standard curve is finally determined to be 1×10 7 ~1×10 2 copies / μL, 10-fold serial dilution; make corresponding light chain standard DNA templates of different concentrations;

[0071] The total volume of the light chain standard reaction is 20 μL, including Taqman Universal Master Mix Ⅱ 10 μL; VEGFLF (10 μM) 0.25 μL; VEGFLR (10 μM) 0.25 μL; VEGFLP probe (SEQ ID NO: 3) 0.5 μL; sterile ultrapure water 8 μL ; Light chain standard DNA template 1 μL;

[0072] The reaction conditions were 50°C for 2min; 95°C for 10min; 95°C for 15s and 60°C for 1min, a total of 40 cycles.

[0073] qPCR amplification map such as figure 1 As shown...

Embodiment 3

[0075] Example 3 Quantitative detection of light chain genes

[0076] Extract the genomic DNA of the cells: CHO cells transfected with the VEGF antibody gene cultured to the 1st, 5th, 10th, 15th, 20th, 25th and 30th generations were divided into approximately 3×10 6 Collect each cell / branch, centrifuge to remove the supernatant, and store the precipitate in a -80°C refrigerator for later use;

[0077] According to the instructions of Purelink Genomic DNA kits, the genomic DNA of the above passage cells was extracted, the concentration was measured with a BioPhotometer plus nucleic acid and protein analyzer, and the genomic DNA was diluted to 1 μg / mL with sterile ultrapure water as a qPCR test sample (for Test product), sterile ultrapure water as NTC control;

[0078] The reaction system and reaction conditions were the same as those in Example 2, and the standard samples and test samples in Examples 2 and 3 were reacted synchronously using the same instrument (Applied Biosyst...

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Abstract

The invention provides a primer probe composition for detecting cell genetic stability and application thereof. The primer probe composition comprises a primer pair SEQ ID NO:1-2 of a VEGF light chain gene and a probe SEQ ID NO:3, and further comprises a primer pair SEQ ID NO:4-5 of a VEGF heavy chain gene and a probe SEQ ID NO:6. According to the primer probe composition, fluorescent quantitative PCR based on a Taqman probe method is adopted for quantitative detection of antibody light and heavy chain gene copy numbers in cells of different culture generations, cell genome DNA is extracted and PCR amplification is carried out, amplification products are used for preparation of target gene standard substances, and specificity and accuracy of quantitative detection of the gene copy numbers are substantially improved.

Description

technical field [0001] The invention belongs to the field of biological technology and the technical field of gene detection, and relates to a primer probe composition for detecting the genetic stability of cells and its application, in particular to a primer probe composition and a reagent for detecting the genetic stability of recombinant CHO cells Boxes, detection methods and their applications. Background technique [0002] Taqman probe method is a widely used fluorescent quantitative PCR analysis method. Compared with ordinary PCR, the PCR reaction system based on Taqman probe not only includes primers, dNTPs, DNA polymerase and template, but also adds probes with fluorescent groups and quenching groups at both ends, with a length of about 20-25 nt . This method utilizes the 5'-3' exonuclease property of DNA polymerase. During the extension process after PCR annealing, the DNA polymerase gradually cuts off the probe, and the fluorescent group at the 5' end and the que...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6876C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6876C12Q2563/107C12Q2531/113
Inventor 连晓宁刘西燕王海燕朱文娟徐瑞君刘军黄纯莹
Owner TOT BIOPHARM CO LTD
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