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Method for detecting similarity of oligonucleotide and target genome

An oligonucleotide and genome technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of unavailability, high cost, cumbersome operation, etc., achieving low cost, simple operation, and time-consuming less effect

Active Publication Date: 2012-10-17
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is very expensive and cumbersome to operate, and cannot be truly applied.

Method used

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  • Method for detecting similarity of oligonucleotide and target genome
  • Method for detecting similarity of oligonucleotide and target genome
  • Method for detecting similarity of oligonucleotide and target genome

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1, the method for detecting oligonucleotide of the present invention and human genome and transcriptome sequence similarity

[0048] 1. Raw materials

[0049] 1. Oligonucleotides to be tested: 4 newly designed tags with unknown similarity are: tag1, tag2, tag3, tag4; ​​the tag with known high similarity is the primer of human GADPH gene; the tag with known low similarity is currently widely used tag2003. details as follows:

[0050] Table 2, Tag

[0051]

[0052] 2. The human genome DNA and total RNA mixture is human leukocyte genome DNA or human leukocyte total RNA.

[0053] 3. Random primer (9N random primer): the sequence is NNNNNNNNNN.

[0054] 4. Artificially synthesized 9N random primers (tag-9N) with Tags shown in Table 2 above attached to their 5' ends.

[0055] 2. Method

[0056] Nucleic acid purification kit (MN company), which contains nucleic acid binding buffer, nucleic acid purification column, washing solution and eluent.

[0057] Kle...

Embodiment 2

[0116] Embodiment 2, the detection method of prior art

[0117] 1. Raw materials

[0118] 1. Artificially synthesized oligonucleotides to be tested that are labeled with fluorescein TAMRA at the 5' end, among which the four newly designed tags with unknown similarity are: tag1, tag2, tag3, tag4; ​​tags with known high similarity It is the primer of human GADPH gene; the known tag with low similarity is currently widely used tag2003. details as follows:

[0119] Table 2, Tag

[0120]

[0121]

[0122] 2. A gene chip with human genomic DNA (customized by Boao Biological Co., Ltd.).

[0123] 3. Hybridization buffer (purchased from Boao Biological Co., Ltd.)

[0124] 2. Method

[0125] Take 1nmol of the artificially synthesized oligonucleotide to be tested that is labeled with fluorescein TAMRA at the 5' end, dilute it with water to 7.5 microliters, then add 7.5 microliters of hybridization buffer, and heat denature at 95°C for 3 minutes (in a PCR machine) , quenched i...

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Abstract

The invention discloses a method for detecting the similarity of an oligonucleotide and a target genome. The method provided by the invention comprises the steps that: (1) a target genome DNA is adopted as a template, a random primer with a 5' terminal connected with the oligonucleotide to be tested is adopted as a primer, and a DNA synthesizing reaction is carried out, such that a double-chain DNA with a 5' terminal of one chain bearing the oligonucleotide A is obtained, and is marked as a product I; (2) the product I is adopted as a template, the oligonucleotide to be tested is adopted as a primer, and a PCR amplification reaction is carried out. If no PCR amplification product is obtained, it is determined that the oligonucleotide has no similarity with the target gene. If a PCR amplification product is obtained, it is determined that the oligonucleotide has similarity with the target gene. The PCR amplification product is a non-primer-dimer. As a result of experiments, the method provided by the invention can be used for accurately and visually detecting the similarity of a tag sequence and a certain biological genome. Also, the method is advantaged in simple operation, low cost, and low time consumption.

Description

technical field [0001] The invention relates to a method for detecting the similarity between an oligonucleotide and a target genome or transcriptome. Background technique [0002] Oligonucleotide tags that have no similarity (homology) to known biological genome or transcriptome sequences play an increasingly important role in modern molecular biology applications. For example, in RNA template-specific PCR amplification (RS-PCR), a reverse transcription primer with an oligonucleotide tag at the 5' end is used in reverse transcription. Finally, using this oligonucleotide label as a primer for PCR amplification, only the reverse transcription product of RNA can be amplified without amplifying the contaminated genomic DNA, so as to realize the specific PCR amplification of the RNA template and avoid the occurrence of false positives. Wherein, the oligonucleotide tags used must have no sequence similarity with the genome or transcriptome of the organism. [0003] In SNP geno...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张岩孙义民杨萍张亮
Owner CAPITALBIO CORP
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