Localised Rca-Based Amplification Method Using A Padlock-Probe

A local, product technology, applied in the conceptual field of local highly linear amplification, to achieve the effects of enhanced signal amplification, enhanced sensitivity, and fast signal generation

Inactive Publication Date: 2016-09-28
欧凌科生物科技公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Although methods for signal amplification based on performing a secondary amplification of the initial RCA product have been described, there are enhanced sensitivity and / or performance for development, such as stronger signal, faster signal generation and / or Ongoing need for assays with improved signal-to-noise ratios

Method used

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  • Localised Rca-Based Amplification Method Using A Padlock-Probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0197] This example investigates linkage and efficiency and effects of different lock:RCA monomer ratios.

[0198] The approximate experimental setup is as follows: The first RCA is generated by using a set of dNTPs containing dUTP. Different lock concentrations and linker components are then added. After ligation, the reaction mixture was treated with UNG or UNG+Exonuclease I / Ill to degrade the first RCA product or the first RCA product and any unligated linear locks. Samples were then analyzed on 6% TBE-urea gels.

[0199] Several different amounts of pre-generated uracil-containing 1st RCA products were added to 50 μl 2nd lock ligation mix (which consisted of 1X buffer 4 (New England Biolabs), 0.2 mM ATP, 0.02 U T4 DNA ligase (Thermo -Scientific) and 0.02mg / ml BSA (New England Biolabs) and 100nM p-bx locked oligonucleotide (SEQ ID No.11) composition) to obtain the first 3:9, 3:1 or 3:0.02 2RCA lock: the first RCA monomer. Reactions were incubated at 37°C for 30 minutes....

Embodiment 2

[0201] stability test

[0202] This example was performed to test the stability of the lock sRCA at high temperature ex 65°C and 95°C. 1 μl of the first RCA product was added to 50 μl of the second lock ligation mix (made with 1X buffer 4 (New England Biolabs), 0.2 mM ATP, 0.02 U T4 DNA ligase (Thermo-Scientific) and 0.02 mg / ml BSA (New EnglandBiolabs) and 100nM p-bx locked oligonucleotide (SEQ ID NO.11) composition). Reactions were incubated at 37°C for 30 minutes. Then 1 mM dNTP, 300 nM sRCA-x-primer oligonucleotide and 200 nM sRCA-d-1b-Cy3 oligonucleotide and 0.1 U phi29 polymerase (Thermo-Scientific) were added to the reaction mixture, followed by incubation at 37° C. Incubate for 100 minutes. The reaction mixture was then divided into 5 parts, and each part was subjected to different heat treatment conditions. 10 [mu]l of each heated sample was then placed on a poly-L-lysine slide and visualized with an EPI fluorescence microscope (Carl Zeiss). The results are show...

Embodiment 3

[0204] Experiments compared the results of different rounds of RCA using padlock probes and showed the signal amplification achievable with padlocked sRCA; stronger signals were seen for 2 (2RCA) or 3 (3RCA) rounds of RCA.

[0205]Streptavidin-coated glass slides (Surmodics) were incubated with 50 μl of 1 pM primary lock ligation mixture for 30 minutes at 37° C., followed by 2 washes with 50 μl of 1 XPBS + 0.05% Tween 20. 50 μl of RCA mix (consisting of 1X Buffer 4 (New England Biolabs), 1 mM dNTPs, 0.1 U phi29 polymerase (Thermo-Scientific), 0.02 mg / ml BSA (New England Biolabs)) was then applied to the slide, And incubated at 37° C. for 20 minutes, then washed twice with 50 μl of 1XPBS+0.05% Tween 20. Afterwards, 50 μl of ligation mix (made of 1X buffer 4 (New England Biolabs), 0.2 mM ATP, 0.02 U T4 DNA ligase (Thermo-Scientific) and 0.02 mg / ml BSA (New England Biolabs) and 100 nM p-bx Locked oligonucleotide (SEQ ID No. 11) was applied to the glass slide and incubated at 37°...

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Abstract

The present invention provides a method for performing a localised RCA reaction comprising at least two rounds of RCA, wherein the product of a second RCA reaction is attached, and hence localised, to a product of a first RCA reaction, said method comprising: (a) providing a concatemeric first RCA product comprising repeated monomers; (b) directly or indirectly hybridising to monomers of said first RCA product a circularisable oligonucleotide comprising target-complementary 3' and 5' end regions such that the 3' and 5' ends of said oligonucleotide hybridise in juxtaposition for ligation directly or indirectly to each other, wherein the target is a sequence in a monomer of said first RCA product or an intermediate molecule hybridised thereto, and wherein the target-complementary endregions of said circularisable oligonucleotide are 6 to 16 nucleotides in length; (c) directly or indirectly ligating the ends of said circularisable oligonucleotide to circularise the oligonucleotide, thereby to provide a template for a second RCA reaction, wherein when said ends are indirectly ligated (i) either a gap oligonucleotide is provided which hybridises to the monomers of the first RCA product in between the 3' and 5' ends of the circularisable oligonucleotide such that it may be ligated to the respective ends, or the hybridised 3' end of the circularisable oligonucleotide is extended by a polymerase such that the extended 3' end may be ligated to the hybridised 5' end, and (ii) the total length of the region of the second RCA template directly or indirectly hybridised to the monomers is no longer than 32 nucleotides in length; and (d) performing a second RCA reaction using said second RCA template of (c) and a primer for said second RCA, to form a second RCA product, wherein in said second RCA reaction the second RCA template remains attached to the first RCA product, and thereby the second RCA product is attached to the first RCA product.

Description

technical field [0001] The present invention generally belongs to the field of nucleic acid amplification using rolling circle amplification (RCA), and in particular to the concept of producing RCA-based locally highly linear amplification in at least two rounds of amplification. The product of the second (or further) RCA reaction is not physically derived from the first (or earlier) RCA product, and the second or further round of RCA amplification provides The way in which the signal obtained by the amplification reaction is amplified. The method of the invention comprises generating a second RCA product by RCA of a second separate RCA template. The second RCA template is provided by a so-called padlock probe, which is a circularisable oligonucleotide so that the ends of the oligonucleotide can be directly or indirectly ligated to each other to form a The circularized oligonucleotides of the RCA products are topologically linked (in strands or "chains") to hybridize to the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6804C12Q1/6809C12Q1/6816C12Q1/6853C12Q1/6862C12Q1/6844C12Q2531/125C12Q2563/179C12Q2531/143C12Q2537/125C12Q2531/119C12Q2537/143C12Q2539/113C12Q2525/161C12Q2565/102C12Q2525/307C12Q2563/131C12Q1/6876
Inventor 乌尔夫·兰德格伦陈磊
Owner 欧凌科生物科技公司
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