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Double fluorescent quantitative PCR kit used for detecting gene expression

A dual fluorescence and gene expression technology, applied in fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve troublesome problems and achieve the effect of saving the amount of reagents and samples

Active Publication Date: 2012-05-30
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, double fluorescent quantitative PCR is used to detect gene expression changes. Usually, it is necessary to re-select internal reference genes and design primers and probes for internal reference genes every time a gene is detected, which is very troublesome.

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  • Double fluorescent quantitative PCR kit used for detecting gene expression
  • Double fluorescent quantitative PCR kit used for detecting gene expression
  • Double fluorescent quantitative PCR kit used for detecting gene expression

Examples

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example 1

[0028] One, the composition of kit of the present invention

[0029] 1. 2×TaqMan universal PCR reaction solution (2×TaqMan universal PCR Mastermix, purchased from ABI Company in the United States): Contains polymerase and other components necessary for PCR reactions, except templates, primers and probes;

[0030]2. The standard product is Bcr-ablP210 T-A vector, which is a control plasmid vector carrying bcr-ablP210 and the fragment of the internal reference gene abl at the same time. Its preparation method is as follows:

[0031] Prepare the following DNA solutions in a microcentrifuge tube, the total amount is 5 μl (Table 1);

[0032] Table 1

[0033]

[0034] 2) Add 5 μl (equal volume) of Solution I;

[0035] 3) React at 16°C for 30 minutes.

[0036] Note: ①The ligation reaction can be carried out normally at room temperature (25°C), but the reaction efficiency is slightly reduced.

[0037] ②The ligation reaction can be carried out normally even after 5 minutes, but ...

example 2

[0089] One, the composition of kit of the present invention:

[0090] 2×TaqMan PCR Universal Reaction Solution (2×TaqMan universal PCR Mastermix, purchased from ABI Company in the United States): Contains polymerase and other components necessary for PCR reactions, except templates, primers and probes;

[0091] The standard is Bcr-abl P190 T-A carrier, which carries bcr-abl at the same time P190 And the control plasmid vector of internal reference gene abl, its preparation method is as follows:

[0092] 1) Prepare the following DNA solutions in a microcentrifuge tube according to Table 2, the total volume is 5 μl.

[0093] table 3

[0094]

[0095] 2) Add 5 μl (equal volume) of Solution I.

[0096] 3) React at 16°C for 30 minutes.

[0097] Note) ①The ligation reaction can be performed normally even at room temperature (25°C), but the reaction efficiency is slightly lower.

[0098] ②The ligation reaction can be carried out normally even after 5 minutes, but the reactio...

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Abstract

The invention relates to a double fluorescent quantitative PCR kit used for detecting gene expression. The kit comprises PCR reaction liquid, DNA polymerase, a proof sample, a primer, a fluorescent probe, a primer and a fluorescent probe, wherein the primers and the fluorescent probes are used for detecting a target gene and an internal reference gene. The kit is characterized in that the sequence of an upstream primer of the internal reference gene which is an abl for detecting the gene is 5'-GGG CGG CCT GAA TGA AG-3', the sequence of a downstream primer is 5'-GCG CTG AAC AAGTTG GTC TTT-3', and the sequence of the fluorescent probe is 5'-TGA GCG CCT TCT CCC CAA AGA-3'; the 5' end of the fluorescent probe is marked with a fluorescent base group, and the 3' end is marked with a quenching base group. The kit can reach the detection accuracy and sensitivity of the prior single fluorescent quantitative PCR method, but the required sample quantity and the required reagent quantity are saved by nearly half.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a quantitative detection method for gene expression. Background technique [0002] Real-time fluorescence quantitative PCR technology was launched by Applied Biosystems in 1996. Because this technology not only realized the leap from qualitative to quantitative PCR, but also compared with conventional PCR, it has stronger specificity, higher degree of automation, and effective solution. It has been widely used in the fields of medicine and food testing due to its characteristics such as PCR contamination. [0003] The reaction system of fluorescent quantitative PCR is composed of a pair of primers (upstream primer P1 and downstream primer P2) and a probe T (such as figure 1 ), the probe is labeled with a fluorescent group R and a quencher group Q. Before the reaction, the fluorescent signal on the probe does not emit light due to the existence of the quenching group. When the reacti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 江千里江汕孟凡义阴常欣李乐戴敏张洋常威周红升李清徐波儿郑潇潇
Owner SOUTHERN MEDICAL UNIVERSITY
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