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Padlock probe detection method

A padlock probe and probe technology, applied in the field of detection of analytes, can solve problems such as extension product amplification

Pending Publication Date: 2019-08-02
Q LINEA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Undesired extension products generated in this way can themselves serve as templates for further undesired extension reactions and thus actually represent undesired HRCA reactions leading to amplification of undesired extension products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0244] Example 1 - Template independent signal generation using padlock probes

[0245] Assess the template-independent signal in the assay and use et al., 2012PLoS One 7, e31068 for comparison. Multiplex library of 90 locks complementary to different target nucleic acids in bacteria and yeast with TE buffer (10mM Tris-HCl pH 7.5 1mM EDTA); Dynabeads MyOne TM Streptavidin T1 beads (Invitrogen); used with multiplexed libraries of capture oligonucleotides complementary to different portions of different target nucleic acids.

[0246] In each reaction, there were two positive control oligonucleotides: a linear padlock probe with added synthetic template as a control for ligation and RCA reactions; and a preformed loop alone as a control for RCA reactions.

[0247] Since the target nucleic acid is not present in the sample, the signal is not expected and any signal generated is caused by non-specificity between the padlock probe and another nucleic acid molecule (e.g., the su...

Embodiment 2

[0259] Example 2 - Analyte detection

[0260] Evaluate the signal-to-noise ratio, and use et al., 2012PLoS One 7, e31068 for comparison.

[0261] Experiments were performed as above, where 1250 copies of genomic E. coli DNA were added to positive reactions. Negative controls were performed as described above. exist Figure 4 The signal and signal-to-noise ratio values ​​generated for each positive and negative reaction are shown in .

[0262] The data show that the signal generated in the positive control sample using the method of the present invention is equivalent to the signal generated by the prior art method, but the signal generated in the negative control sample is lower (ie, reduced background) using the new method of the present invention.

[0263] It should be noted that the negative control in the first experiment performed using the prior art method had an unexpectedly high level of background signal. This indicates one of the effects addressed by the pres...

Embodiment 3

[0264] Example 3 - Combined RO / DO Sequence

[0265]Padlock probes with individual array oligonucleotide sequences and combined detection and restriction oligonucleotide sequences are used in the methods described above. A total of 90 padlock probes are used in the padlock probe library.

[0266] Samples of genomic DNA preparations spiked with four bacterial reagents, one of which also contained the sequence of the resistance gene mecA, were analyzed at input amounts of 2500, 500 or 100 genome copies, and the signal was compared to that obtained in the negative control signals for comparison.

[0267] Measure the signal-to-noise ratio of each reaction, and as Figure 5 shown. Signal-to-noise ratios between 10 and 100 were achieved in 100 genome copies.

[0268] The background signal was 47, which was reported as relative fluorescence units (RFU) in the negative control of the five padlock probe sets reported. The average background signal of the padlock probes remaining ...

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Abstract

The present invention relates to multiplexed methods of detecting an analyte in a sample using two or more padlock probes each specific to a different target sequence, the target sequence being eitherpart of an analyte or indicative of the presence of an analyte in the sample, wherein each padlock probe comprises an analyte-specific reporter sequence, and either a restriction cleavage site located 3' of the analyte-specific reporter sequence, and / or a first amplification primer binding site for an amplification reaction, wherein where the padlock probe comprises a restriction cleavage site, cleavage at said restriction cleavage site occurs 3' of the analyte-specific reporter sequence, and where the padlock probe comprises a first amplification primer binding site for the further amplification reaction, it does not contain a second amplification primer binding site 5' of the analyte-specific reporter sequence, and panels of probes and kits for the same.

Description

technical field [0001] The present invention relates to the field of detection of analytes by rolling circle amplification (RCA), and in particular to improved methods of detecting analytes in samples using padlock probes. Background technique [0002] Rolling circle replication (RCR) is a mechanism for replicating circular DNA molecules such as plasmids or viruses. This reaction has been used as the basis for laboratory methods for the amplification of circular molecules, and has utility in methods for amplifying or generating nucleic acids, where it has been shown to be useful in a variety of methods that use or generate circular nucleic acid molecules as reporters. In this assay; in this assay method, a circular molecule is amplified (replicated) by RCA, and the replicated or amplified circular nucleic acid molecule is detected. In other methods, desired or target molecules can be circularized and amplified by RCA. Accordingly, rolling circle replication (RCR) is now co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6853C12Q1/6823
CPCC12Q1/6823C12Q1/6853C12Q2525/131C12Q2525/155C12Q2525/307C12Q2531/125C12Q2533/107C12Q2537/143C12Q2561/125C12Q2563/179C12Q1/682
Inventor 卡米拉·罗素杰尼·约兰松马茨·格尔伯格
Owner Q LINEA AB
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