HER2 gene fluorescent in-situ hybridization probe as well as preparation method and application thereof

A fluorescence in situ hybridization and probe technology, applied in biochemical equipment and methods, DNA preparation, DNA/RNA fragments, etc., can solve the problem of uncertain number of fluorescent probes, low probe specificity, and lack of small fragmentation etc. to achieve the effects of reducing background interference, increasing hybridization efficiency, and reducing hybridization time

Active Publication Date: 2019-01-08

广州简册生物技术有限公司

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AI-Extracted Technical Summary

Problems solved by technology

However, there are some non-specific sequences in the BAC and PCR product probes, the probe specificity is not high, and the signal background is high

Existing probe fragments are generally too large, generally between 200-500nt in length, lack of effective methods for s...

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Method used

Carry out single amplification by using emulsion PCR, amplified product transcription builds library RNA, then constructs and obtains single-stranded DNA probe by library RNA reverse transcription, directly carries out PCR amplification and obtains by probe library compared with tradition Double-stranded DNA probes can make full use of transcription and reverse transcription to obtain linear single-stranded DNA. During the amplification process of the probe library, there will be no non-specific amplification, reducing biased amplification, and improving the efficiency of the probe. Uniformity, which in turn helps to improve the accuracy of subsequent in situ hybridization.

For different target gene regions, construct candida...

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Abstract

The invention discloses an HER2 gene fluorescent in-situ hybridization probe as well as a preparation method and application thereof. The preparation method of the HER2 gene fluorescent in-situ hybridization probe as well as the HER2 gene fluorescent in-situ hybridization probe prepared with the preparation method are specially designed for a genome non-repeated region, so that non-specific reaction can be reduced and background interference can be reduced; the constructed probe is single-chain DNA, and can reduce pairing of the probe and the own and improve the hybridization efficiency compared with the traditional double-chain probe such as BAC or PCR products; furthermore, the constructed single-chain DNA probe has a small segment, can be combined with a targeted sequence rapidly and can reduce the hybridization time.

Application Domain

Microbiological testing/measurementDNA preparation +1

Technology Topic

FluorescenceIn situ hybridization +7

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Examples

Experimental program(1)

Example Embodiment

[0029] The preparation method of the HER2 gene fluorescent in situ hybridization probe of one embodiment includes the following steps:

[0030] Step 1: Construct the probe library: select the non-repetitive target gene region on chromosome 17 of the human genome containing the HER2 gene, and construct a 35nt-200nt single-stranded fragment that is completely complementary to the target gene region every 1-10bp as Candidate probes, through BLAST comparison, obtain the Tm value of non-specific hybridization, remove the non-specific Tm value for candidate probes at 45°C, and select non-overlapping candidate probes, and select the candidate probes at both ends of the screen. Add the amplified primer fragments to obtain the target probe library.

[0031] The target gene region in this embodiment is a non-repetitive region of the genome. By designing candidate probes for the non-repetitive region, non-specific reactions and background interference can be reduced.

[0032] For different target gene regions, candidate probes are constructed at intervals of a preset length. For example, candidate probes can be constructed at intervals of 1-10 bp, which can avoid mutual interference between probes and help improve the accuracy of detection results.

[0033] Preferably, the length of the candidate probe is preferably between 35-100 nt, more preferably around 45 nt, such as between 40-50 nt. The single-stranded DNA probe constructed in this embodiment has small fragments and can bind to the target sequence more quickly, reducing the hybridization time.

[0034] The amplification primer fragment can be a variety of universal amplification primers. For example, in a specific embodiment, the sequence of the amplification primer fragment is as SEQ ID NO. 1 (5'-GGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGA-3') and SEQ ID NO. 2 ( 5'-CGTGGTCGCGTCTCA-3').

[0035] The probe library constructed in this embodiment uses chip synthesis technology to synthesize the target probe library on a gene chip.

[0036] Step 2: Amplify the target probe library.

[0037] Specifically, this embodiment uses the emulsion PCR amplification method to amplify the target probe library. And in the emulsion PCR amplification, the prepared PCR reaction system is divided into multiple parts, and the PCR reaction is performed on the thermal cycler respectively, and after the reaction is completed, part or all of the PCR products are combined for subsequent processing. The PCR reaction can be carried out but not limited to 25-35 cycles, which can be determined according to the length of the specific probe.

[0038] Step 3: Perform in vitro transcription of the amplified product to prepare library RNA corresponding to the target probe library.

[0039] Step 4: Perform reverse transcription on the library RNA to prepare a single-stranded DNA probe.

[0040] In this embodiment, when the library RNA is reverse transcribed to prepare a single-stranded DNA probe, it includes the step of adding an amino (Amino) dNTP to the reaction system to label the probe, and the resulting single-stranded DNA probe Labeled with amino. In a specific embodiment, amino dUTP (Amino-dUTP) is used for probe labeling. In other embodiments, it is not limited to using amino dUTP for labeling, and amino dATP, amino dCTP, etc. may also be used for labeling.

[0041] The single-stranded DNA probe is constructed by using emulsion PCR for single-stranded amplification, the amplified product is transcribed to construct the library RNA, and then the single-stranded DNA probe is constructed by reverse transcription from the library RNA, compared with the traditional direct PCR amplification of the probe library to obtain double-stranded DNA The probe can make full use of transcription and reverse transcription to obtain linear single-stranded DNA. During the amplification of the probe library, there will be no non-specific amplification, which reduces the biased amplification and improves the uniformity of the probe. This will help improve the accuracy of subsequent in situ hybridization.

[0042] Step 5: Fluorescently label the prepared single-stranded DNA probe to obtain it.

[0043] Correspondingly, when the prepared single-stranded DNA probe is fluorescently labeled, N-hydroxysuccinimide ester (NHS ester) labeled with a fluorescent dye is reacted with an amino-labeled single-stranded DNA probe to react to the single-stranded DNA probe. The DNA probe is fluorescently labeled. The fluorescent dye may be, but is not limited to, the green fluorescent dye Alexa Fluor 488.

[0044] By using reverse transcription to incorporate amino-dNTPs and then coupling with fluorescent dyes, the number of fluorescent markers for each probe can be increased, which is beneficial to increase the fluorescence intensity during fluorescence in situ hybridization and improve the accuracy and reliability of detection results.

[0045] In addition, this embodiment also provides a HER2 gene fluorescent in situ hybridization probe, which is prepared by the preparation method of the HER2 gene fluorescent in situ hybridization probe described in any of the above embodiments, and further provides a HER2 gene Gene fluorescence in situ hybridization detection chip or detection kit, which contains the above-mentioned HER2 gene fluorescence in situ hybridization probe.

[0046] The preparation method of the HER2 gene fluorescent in situ hybridization probe and the HER2 gene fluorescent in situ hybridization probe prepared by the preparation method are single-stranded DNA. Compared with the traditional double-stranded probes such as BAC or PCR products, the detection can be reduced. The pairing of the needle with itself increases the hybridization efficiency; and the constructed single-stranded DNA probe has a small fragment, which can be combined with the target sequence more quickly, reducing the hybridization time.

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HER2 gene fluorescent in-situ hybridization probe as well as preparation method and application thereof

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

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Examples

Example Embodiment

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Description & Claims & Application Information

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