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Fluorescence probe for hgih temperature polyase exonuclease activity in real time PCR test

An exonuclease and fluorescent probe technology, applied in the field of fluorescent probes, can solve the problems of specific amplification signal interference and achieve the effect of improving performance

Active Publication Date: 2007-01-24
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] We found that fluorescent probes with secondary structures, such as displacement probes and molecular beacons, are also substrates for the 3′→5′ excision activity of high-temperature polymerases, which will generate non-specific signals in real-time PCR, causing adverse effects on Interference of specific amplification signal

Method used

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  • Fluorescence probe for hgih temperature polyase exonuclease activity in real time PCR test
  • Fluorescence probe for hgih temperature polyase exonuclease activity in real time PCR test
  • Fluorescence probe for hgih temperature polyase exonuclease activity in real time PCR test

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: AmpliTaq Gold TM The amyloid precursor protein gene was amplified by DNA polymerase and detected in real time with an unmodified displacement probe and a positive-strand 5′ phosphorothioate-modified displacement probe.

[0037] The amyloid precursor protein (APP) gene is located on human chromosome 21. The abnormal expression of this gene is related to some diseases such as Down syndrome and Alzheimer's disease.

[0038] In this embodiment, the APP gene is the target gene, and primers and target-specific replacement probes are designed for real-time PCR detection. The primers used are Primer1 and Primer2, the length of the amplified product fragment is 100bp, and the probe used for detection is Probe1 or Probe2 (see the table for the sequence. 1, the same below).

[0039] 2.5μL of 10XPCR Gold buffer in 25μL reaction solution, 2.0mM MgCl 2 , 200 μM dNTPs, 1.0 U AmpliTaqGold TM DNA polymerase, 10 pmol upstream primer, 10 pmol downstream primer (see Table 1...

Embodiment 2

[0043] Example 2: pfuDNA polymerase amplifies the amyloid precursor protein gene, and detects in real time with unmodified displacement probes and phosphorothioate-modified displacement probes at the negative strand 3' end.

[0044] The target gene and primers in this example are the same as in Example 1, and the probe used for detection is Probe1 or Probe3.

[0045] 25μL reaction solution containing 10×Pfu reaction buffer (containing 20mM MgCl 2 ) 2.5 μL, 200 μM dNTP, 1.0 Upfu DNA polymerase, 10 pmol upstream primer, 10 pmol downstream primer, 10 pmol probe1 or probe3, 5 μL PCR product dilution template or ultrapure water (negative control). Reaction conditions: 95°C for 3 minutes, cycle period of 94°C for 15s, 55°C for 20s, 72°C for 20s, a total of 40 cycles, each time the fluorescence data of the FAM channel was collected in real time during annealing. MX 3000P real-time PCR instrument was used for detection.

[0046] For real-time PCR results, see figure 2 , the replacem...

Embodiment 3

[0047] Example 3: EX Taq DNA polymerase amplifies the amyloid precursor protein gene, using unmodified displacement probes, 5' and 3' phosphorothioate-modified displacement probes for long-fragment real-time PCR amplification detection.

[0048] In this example, the APP gene is the target gene, the primers Primer3 and Primer4 are used, and the length of the amplified product fragment is 1082 bp, and the probe used for detection is Probe1 or Probe4.

[0049] 2.5 μL of 10×EX Taq reaction buffer in 25 μL reaction solution, 2.0 mM MgCl 2 , 200 μM dNTP, 1.0 UEX Taq enzyme, 10 pmol upstream primer, 10 pmol downstream primer, 10 pmol probe1 or probe4, 5 μL genome template or ultrapure water (negative control). Reaction conditions: 95°C for 3 minutes, the cycle period is 94°C for 15s, 55°C for 30s, and 72°C for 45s, a total of 40 cycles, each time the fluorescence data of the FAM channel is collected in real time during annealing. MX 3000P real-time PCR instrument was used for detect...

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Abstract

The present invention relates to fluorescent probe, and is especially fluorescent probe for real-time PCR detection and capable of resisting high temperature polyase exonuclease activity. The fluorescent probe has two-stage structure containing complementary sequences and including replacing probe, molecular beacon probe, scorpion primer, etc. The fluorescent probe includes a fluorescent probe such modified that it can resist the 5'-->3' exonuclease activity of high temperature polyase without generating non-specific signal, a fluorescent probe such modified that it can resist the 3'-->5' exonuclease activity of high temperature polyase without generating non-specific signal in real-time PCR detection, and a fluorescent probe such modified that it can resist both 5'-->3' exonuclease activity and 3'-->5' exonuclease activity of high temperature polyase without generating non-specific signal.

Description

technical field [0001] The invention relates to a class of fluorescent probes, in particular to a class of fluorescent probes capable of resisting high-temperature polymerase exonuclease activity in real-time polymerase chain reaction detection. Background technique [0002] Real-time polymerase chain reaction (Real-time PCR, referred to as real-time PCR) means that amplification and detection are carried out simultaneously, and the nucleic acid amplification process is indicated by detecting the change of the fluorescent signal in the amplification cycle. [0003] Like conventional PCR, real-time PCR requires the use of high-temperature polymerases for amplification. In most cases, real-time PCR uses the common high-temperature polymerase Taq. In addition to polymerase activity, Taq enzyme also has 5'→3'exonuclease activity. In a few cases, people will use Taq that has been genetically modified to remove the 5'→3' exonuclease activity, such as KlenTaq, etc. When high-fid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/25
Inventor 黄秋英李庆阁
Owner XIAMEN UNIV
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