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RCA-based local amplification method
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A local and probe technology, applied in the field of nucleic acid amplification, to achieve the effects of fast signal generation, enhanced signal amplification, and rapid determination
Active Publication Date: 2018-09-25
欧凌科生物科技公司
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[0019] Although methods for signal amplification based on performing a second 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
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[0417] use figure 1 The probe depicted in exemplifies the sRCA principle.
[0418] Generation of probes
[0419] The first and second RCA templates are pre-ligated separately in solution to form the RCA "probe" (primer / template complex). 300 nM RCA template, 100 nM RCA primer and ligation buffer (1X T4 ligase buffer, 1 mM ATP and 0.05 U / μL T4 ligase) were mixed at 37° C. for 30 minutes.
[0420] Immobilization of the first RCA probe
[0421] A 100 fM dilution of the first RCA probe was prepared in 1XPBS. 50 μL of the 100 fM dilution was applied to streptavidin-coated slides (Codelink) for 30 minutes at room temperature. After incubation, slides were washed twice with wash buffer (1XPBS, 0.05% Tween 20).
[0422] Generation of the first RCA product
[0423] After removing the wash buffer, RCA mix (1X phi29 buffer (Fermentas), 250 μM dNTPs, 0.02 U / μL phi29 polymerase (Fermentas)) was added to the slide and incubated at 37° ...
Embodiment 2
[0430] Example 2 - Circular RCA probes for detection of RCA products
[0431] This example demonstrates the utility of circular RCA probes in super RCA (sRCA) reactions, such as Figure 11 An embodiment of the unfolded RCA reporter probe is depicted.
[0432] Preligated locks were amplified by RCA using phi29 polymerase for 1 hr at 37°C. The circular RCA probe was then incorporated and the reaction was continued at 37°C for 1 hour followed by 10 minutes at 65°C to inactivate the phi29 polymerase. Cy-3 or FITC-labeled oligonucleotides targeting the primary and secondary RCA products, respectively, were then added to the sRCA reaction and allowed to hybridize at 55°C for 20 minutes. sRCA products were adhered to poly-L-lysine-coated slides and subsequently visualized by epifluorescence microscopy using a 20X objective with an exposure time of 1500 ms.
[0433] Figure 21 A shows that the secondary RCA product is not produced in the absence of the initial RCA product. Fig...
Embodiment 3
[0434] Example 3 - Blocking the 3' end of the probe to avoid target-templated extension and subsequent probe transition from the first (initial) Importance of displacement of RCA products
[0435] Streptavidin-coated slides (SurModics) were compartmentalized with secure-Seal 8 (Grace Bio-labs) prior to the experiment. In each reaction chamber, 50 μl of 1 pM synthetic biotinylated DNA template was incubated in 1× PBS at 37° C. for 60 minutes. After incubation, block with blocking buffer (0.1% BSA (Sigma), 100nM goat IgG (Sigma), 1mM biotin (Sigma), 10ng / μl salmon spermDNA (Sigma), 5mM EDTA, 1x PBS and 0.05% Tween 20) Slides were blocked at 37°C for 60 minutes. A wash step (with 2 replicates of 50 μl 1×PBS 0.05% Tween20) was performed before each addition of a new mixture to remove previous incubation reagents.
[0436] Add 50 μl of ligation mix (1x NEB4 buffer (NEB), 0.5 μg / μl BSA (NEB), 15 units of T4 DNA ligase (Fermentas) and 0.5 mM ATP (Fermentas)) and 100 nM of padl...
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Abstract
The present invention provides a method for performing a localized RCA reaction comprising at least two rounds of RCA, wherein the product of the second RCA reaction is attached and thus confined to the product of the first RCA reaction, the method comprising: (a) providing the first RCA product; (b) directly or indirectly hybridizing a probe comprising or providing primers for a second RCA reaction to said first RCA product; and (c) performing a second RCA using said RCA primers of (b) react to form a second RCA product, wherein in said reaction: (i) said probe and said primer cannot initiate extension using said first RCA product as a template, or limit any said extension to avoid interference with said first RCA product replacement of any probes hybridized to the first RCA product; (ii) maintaining direct or indirect hybridization of the RCA primers of (b) to the first RCA product and, by virtue of the hybridization, the second RCA product attached to said first RCA product; (iii) the RCA template for said second RCA reaction is included in or provided by said probe, or provided separately. The method is particularly useful in the detection of an analyte where the analyte is a nucleic acid or where the nucleic acid is used or produced as a marker for the analyte.
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. By ensuring that the reaction product of the second RCA is physically attached to the product of the first RCA reaction, a localized amplification reaction can be obtained. The present invention provides such methods and is particularly useful in the amplification of signal from detection assays based on the detection of RCA product...
Claims
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