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PCR (polymerase chain reaction) nucleic acid detection method

A nucleic acid, technology for nucleic acid, applied in the field of molecular biology

Active Publication Date: 2011-05-25
湖州申科生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection method of Willner and Zhou Xiang is convenient, sensitive, fast and cheap, but it is only suitable for the detection of single-stranded nucleic acid, and has certain limitations in application

Method used

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  • PCR (polymerase chain reaction) nucleic acid detection method
  • PCR (polymerase chain reaction) nucleic acid detection method
  • PCR (polymerase chain reaction) nucleic acid detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, detect DNA T-1 with oligonucleotide probe 1

[0045] For the reaction steps for detecting nucleic acids, see figure 1 . One end of the oligonucleotide probe 1 is labeled with a DNAzyme sequence that combines with porphyrin iron and the like and has peroxidase activity, and the other end is a sequence that can form a partial complementarity with the DNAzyme sequence. Partial complementarity makes the oligonucleotide probe 1 form a stem-loop hairpin structure with a short single strand at one end, and the loop portion of the hairpin structure of the oligonucleotide probe 1 can be complementary to the target nucleic acid sequence DNA T-1.

[0046] After adding oligonucleotide probe 1 to the PCR system, under denaturing conditions, the double strand of the DNA T-1 to be tested and the hairpin structure of probe 1 are opened; under annealing conditions, the forward primer and probe Both hybridize with one strand of DNAT-1; under the action of Taq enzyme, the ...

Embodiment 2

[0067] Embodiment 2, detect DNA T-1 with oligonucleotide probe 2

[0068] For the reaction steps for detecting nucleic acids, see figure 2. Oligonucleotide probe 2 contains two fragments A and B, both of which contain part of the DNAzyme sequence and a complementary sequence. After adding fragment A of oligonucleotide probe 2 to the PCR system, under denaturing conditions, the double strand of DNA T-1 to be tested and the hairpin structure of probe 2 are opened; under the conditions of annealing, the forward primer and Probe 2 hybridizes with one strand of DNAT-1; under the action of nucleic acid polymerase, the forward primer is extended; when extending to probe 2, due to the exonuclease activity of the nucleic acid polymerase from the 5' end to the 3' end , the part of probe 2 hybridized with DNAT-1 is digested and degraded, and fragment C containing part of the DNAzyme is released. At this time, fragment B of oligonucleotide probe 2 is added, and fragment C and fragment ...

Embodiment 3

[0090] Example 3. Detection of Aeromonas hydrophila with oligonucleotide probe 3 (No.: EU678635.1)

[0091] (1) Forward and reverse primers 2, oligonucleotide probe 3, partial sequence of Aeromonas hydrophila

[0092] Forward primer 2: 5'-GCC ATG CCG CGT GTG TGA AG-3'

[0093] Reverse primer 2: 5'-AGC CGG TGC TTC TTC TGC GA-3'

[0094] Oligonucleotide probe 3: 5’-CCC TAC CCA GGG TTG TAA AGC ACT TTC AGC GAG GAG GAA AGG TTG ATG TGG GTA GGG CGG GTT GGG-3’

[0095] Partial sequence of Aeromonas hydrophila: 5′-GCC ATG CCG CGT GTG TGA AGA AGG CCT TCG GGT TGT AAA GCA CTT TCA GCG AGG AGG AAA GGT TGA TGC CTA ATA CGT GTC AAC TGT GAC GTT ACT CGC AGA AGA AGC ACC GGC T-3′

[0096] (2) Reaction system and PCR conditions

[0097] Forward primer 1 μM

[0098] Reverse primer 1 μM

[0099] Taq Enzyme 5U

[0100] dNTPs 0.4mM

[0101] The concentration is 100 / μl Aeromonas hydrophila bacteria liquid 2μl

[0102] 10x Taq buffer 5μl

[0103] Make up the system to 50 μl with ultrapure water...

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Abstract

The invention belongs to the technical field of molecular biology and relates to the analysis and detection of nucleic acids, particularly to a PCR (polymerase chain reaction) detection method which is universal and utilizes 5'-3' excisionenzyme activity of nucleic acid polymerase for releasing DNAzyme. The nucleic acid detection method comprises the following steps: adding an oligonucleotide probe labeled by the DNAzyme or partial DNAzyme in the PCR system, amplifying nucleic acids to be detected by a forward primer and a reverse primer, releasing the DNAzyme label or the partial DNAzyme label in the probe by the 5'-3' excisionenzyme activity of the nucleic acid polymerase, and carrying out qualitative analysis or quantitative analysis of nucleic acids by detecting labels released from PCR products. The method achieves sensitivity, accuracy, fastness and cheapness; and the method can be applied in various gene detection systems and is highly applicable to the early detection and genetic analysis of some diseases, and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to nucleic acid analysis and detection, in particular to a universal PCR detection method for releasing DNAzyme by utilizing the 5'-3' exonuclease activity of nucleic acid polymerase. Background technique [0002] A probe is a molecule that reacts with a specific target molecule and carries a suitable label for detection after the reaction. Nucleic acid probe technology is to use the principle of nucleotide base complementarity to use a specific gene probe, that is, a labeled section of single-stranded DNA (or RNA) molecule that recognizes a specific base sequence, and is complementary to the target sequence to be determined. A technique for detecting the target sequence being tested. At present, nucleic acid probe technology has been widely used in clinical microbiology, blood screening, diagnosis and prevention of genetic diseases, forensic science and other fields, and is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 唐卓杜凤
Owner 湖州申科生物技术股份有限公司
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