Multi-RCA (rolling circle amplification) method based on split padlock probes

A padlock probe and multiplex technology, applied in the field of multiplex RCA based on split padlock probes, can solve the problems of high false negative, high cost, complicated steps, etc., and achieve high specificity, convenient operation and strong specificity. Effect

Inactive Publication Date: 2012-10-10
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages: Electrophoresis after PCR amplification is required, which requires high instrument conditions; the location of mutations cannot be detected; the sensitivity of detection decreases with the increase of DNA fragment length; false negatives are high; the detection rate of AT is not as good as CG, which has limitations
Disadvantages: Only about 1 / 3 of the bases in sequence polymorphic loci involve restriction enzyme recognition sequences, and the range of multi-site detection is limited; restriction enzyme digestion conditions are relatively high, and the steps are cumbersome
Disadvantages: higher cost, longer time-consuming

Method used

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  • Multi-RCA (rolling circle amplification) method based on split padlock probes
  • Multi-RCA (rolling circle amplification) method based on split padlock probes
  • Multi-RCA (rolling circle amplification) method based on split padlock probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Taking isoniazid-resistant clinical isolates of Mycobacterium tuberculosis (katG315 mutation) as an example of the target sequence probe, the specific operation steps are as follows:

[0046] Step 1. Ligation reaction: take 1uL of the genome extract of clinical isolates and 0.1μmol / L split padlock probe (the final concentration of the four probes is 0.1umol / L), the base sequence of which is shown in the sequence table sequence 11, the target sequence Mix with the probe, boil and denature, immediately place it on ice for 5 minutes, take it out, raise the temperature to 37°C, hybridize for 15 minutes, add 2.5U of T4 DNA ligase and 1 μL of T4 DNA ligase buffer, deionized water to make up 10uL of the reaction system, and react The time is 45min, then add 10U exonucleaseI and 10U exonucleaseIII, and react at 37°C for 15min to prepare a ligation product containing a circular template; the composition of the T4DNA ligase buffer is 40mmol / L Tris-HCL, 10mmol / L MgCL 2 ...

Embodiment 2

[0055] Example 2: Taking isoniazid-resistant clinical isolates of Mycobacterium tuberculosis (inhA-15 mutation) as an example of the target sequence probe, the specific operation steps are as follows:

[0056] Step 1. Ligation reaction: Take 1uL of the genome extract of the clinical isolate and 0.1μmol / L split padlock probe (the final concentration of the four probes is 0.1umol / L). Mix with the probe, boil and denature, immediately place it on ice for 5 minutes, take it out, raise the temperature to 37°C, hybridize for 15 minutes, add 2.5U of T4 DNA ligase and 1 μL of T4 DNA ligase buffer, deionized water to make up 10uL of the reaction system, and react The time is 45min, then add 10U exonucleaseI and 10U exonucleaseIII, and react at 37°C for 15min to prepare a ligation product containing a circular template; the composition of the T4DNA ligase buffer is 40mmol / L Tris-HCL, 10mmol / L MgCL 2 ,10mmol / L DTT and 0.5mmol / L ATP;

[0057] Step 2. Rolling circle amplification reaction...

Embodiment 3

[0060] Example 3: Taking rifampicin (RFP) drug-resistant clinical isolates (rpoB531 mutation) of Mycobacterium tuberculosis as an example of the target sequence probe, the specific operation steps are as follows:

[0061]Step 1. Ligation reaction: Take 1uL of the genome extract of the clinical isolate and 0.1μmol / L split padlock probe (the final concentration of the four probes is 0.1umol / L). Mix with the probe, boil and denature, immediately place it on ice for 5 minutes, take it out, raise the temperature to 37°C, hybridize for 15 minutes, add 2.5U of T4 DNA ligase and 1 μL of T4 DNA ligase buffer, deionized water to make up 10uL of the reaction system, and react The time is 45min, then add 10U exonucleaseI and 10U exonucleaseIII, and react at 37°C for 15min to prepare a ligation product containing a circular template; the composition of the T4DNA ligase buffer is 40mmol / L Tris-HCL, 10mmol / L MgCL 2 ,10mmol / L DTT and 0.5mmol / L ATP;

[0062] Step 2. Rolling circle amplificati...

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Abstract

The invention discloses a multi-RCA (rolling circle amplification) method based on split padlock probes. Each novel split padlock probe is about 90bp in length and comprises four parts, namely a detection arm, a universal primer area, an HhaI endonuclease site and a tag sequence area, wherein an amplification system is composed of a connection system and an RCA system. The concrete detection method comprises the steps of: firstly, carrying out coupled reaction, mixing a target sequence DNA (deoxyribonucleic acid) segment and four split padlock probes of which the final concentration is 1mol/L, hybridizing for 15 minutes after boiling and degenerating, adding T4DNA ligase and T4DNA ligase buffer solution, supplying 10 muL of reaction system; carrying out exonuclease I and exonuclease III exterior contact for 45 minutes at 37 DEG C, preparing an annular template and then carrying out RCA reaction; mixing, boiling and degenerating 10 muL of connection product and universal primer, respectively adding dNTP, phi29DNA polymerase, HhaI restriction enzyme and buffer solution to form 20 muL of reaction system, stewing for 60 minutes at 37 DEG C; finally detecting single-chain DNA product of RCA with a specific tag sequence, thus obtaining a corresponding test conclusion according to the result.

Description

technical field [0001] The invention relates to an RCA method of molecular probes, in particular to a multiple RCA method based on split padlock probes. Background technique [0002] Padlock probes, also known as circularizing oligonucleotide probes (OCP), are a sensitive, accurate and specific molecular diagnostic method that can meet the needs of life sciences. Rolling circle amplification (rolling circle amplification, RCA) is an amplification method that imitates the self-replication form of bacteriophage after infection of bacteria. This form of replication can perform relatively unlimited single-stranded amplification of circular single-stranded DNA at constant temperature. . Under certain conditions, we use the circular single-stranded DNA formed by the padlock probe to carry out rolling circle replication to achieve nucleic acid amplification under constant temperature conditions. In recent years, it has been widely used in some gene detection technology research, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 向阳黄庆府伟灵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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