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

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

Example Embodiment

[0044] Example 1. Using oligonucleotide probe 1 to detect DNA T-1

[0045] Refer to the reaction steps for detecting nucleic acid figure 1 . One end of the oligonucleotide probe 1 is labeled with a DNAzyme sequence that binds to porphyrin iron and has peroxidase activity, and the other end is a partially complementary sequence with the DNAzyme sequence. The two ends can be Partially complementary, so that the oligonucleotide probe 1 forms a stem-loop hairpin structure with a small single strand at one end, and the loop of the oligonucleotide probe 1 hairpin structure 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, both the double-stranded DNA T-1 and the hairpin structure of probe 1 are opened; under renaturation conditions, the forward primer and probe Both hybridize with one of the strands of DNAT-1; under the action of Taq enzyme, the forward primer is extended; when ...

Example Embodiment

[0067] Example 2: Detection of DNA T-1 with oligonucleotide probe 2

[0068] Refer to the reaction steps for detecting nucleic acid figure 2 . Oligonucleotide probe 2 contains two fragments A and B, both of which contain a partial DNAzyme sequence and a complementary sequence. After adding fragment A of oligonucleotide probe 2 to the PCR system, under denaturing conditions, the double-stranded DNA T-1 and the hairpin structure of probe 2 are opened; under renaturation conditions, the forward primer and Probe 2 hybridizes to one of the strands of DNAT-1; under the action of nucleic acid polymerase, the forward primer extends; when it extends to probe 2, nucleic acid polymerase has 5'end to 3'end exonuclease activity , The part of probe 2 hybridized with DNAT-1 is digested and degraded, and fragment C containing part of DNAzyme is released. At this time, fragment B of oligonucleotide probe 2 is added, fragment C and fragment B are complementary through a sequence Can form a comp...

Example Embodiment

[0090] Example 3. Detection of Aeromonas hydrophila with Oligonucleotide Probe 3 (Code: EU678635.1)

[0091] (1) Positive and negative primer 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 1μM

[0098] Reverse primer 1μM

[0099] Taq enzyme 5U

[0100] dNTPs 0.4mM

[0101] The concentration is 100 / μl Aeromonas hydrophila bacterial solution 2μl

[0102] 10 times Taq buffer 5μl

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

[0104] The PCR pr...

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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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