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Full genome rolling circle amplification and its product fixing method

A rolling circle amplification and whole genome technology, applied in the field of immobilization, can solve the problems of limited detection sites and incomplete information, and achieve the effects of saving detection costs, accurate information, and saving template preparation time.

Inactive Publication Date: 2007-04-11
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these technologies can complete the detection of mutations or nucleotide polymorphisms to a certain extent, they first need to amplify the target fragments to improve the sensitivity of detection, so the sites they detect are quite limited, so the information obtained is incomplete

Method used

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  • Full genome rolling circle amplification and its product fixing method
  • Full genome rolling circle amplification and its product fixing method

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] Example 1 Yeast whole genome fixation method

[0025] Referring to Figures 1 and 2, the surface of the microwell plate was hydrophobically treated with dichlorodimethylsilane on a flat glass plate. After the micropores were washed with water and dried, they were modified with 5% 3-aminopropyltrimethoxysilane acetone for 1 hour and treated with 5% glutaraldehyde for 2 hours to derive aldehyde active groups on the surface of the carrier.

[0026] On the other hand, the yeast genome is enzymatically cut into fragments of about 500 bases in size, and these fragmented nucleic acid sequences are ligated with a pair of universal oligonucleotides under the action of ligase, one of which is a universal oligonucleotide. Completely complementary to the sequence of the rolling circle amplification primer.

[0027] The fragmented nucleic acid sequences connected by the tethers are connected into circular molecules under the action of ligase, and the amino-modified rolling circle am...

example 2

[0028] Example 2 Human Whole Genome Fixation Method

[0029] Refer to Figures 1 and 2.

[0030] In one aspect, the human genome is enzymatically cleaved (or sonicated) into fragments with a size of 50-1000 bases, and these fragmented nucleic acid sequences are ligated with a pair of universal linkers under the action of ligase, one of which is The universal oligonucleotide molecules are fully complementary to the sequences of the rolling circle amplification primers.

[0031]The fragmented nucleic acid sequences connected by the tethers are connected into circular molecules under the action of ligase, and the rolling circle amplification reaction is carried out after annealing the rolling circle amplification primers modified with acrylamide groups.

[0032] The acrylamide-modified rolling circle amplification reaction is diluted with 3% acrylamide monomer, mixed into a prepolymer solution, and evenly distributed on a clean glass slide, so that each fragmented single nucleic ...

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Abstract

The full genome rolling circle amplification and its product fixing method realizes the simultaneous acquisition of full genome information to save template preparing time greatly, and the simultaneous amplification of the full genome information after fragmentation in single molecule form can raise the detection sensitivity while maintaining the number of single molecules after fragmentation. The genome DNA is enzyme digested or ultrasonically broken into fragments, the genome DNA fragments are connected with connexons to form circle and amplified with the circular molecule to obtain rolling circle amplified genome fragmented nucleic aid sequence. The nucleic aid sequence is diluted and fixed onto the surface of hard carrier chemically to realize the fixation of full genome sequence and constitute full genome DNA chip.

Description

technical field [0001] The invention relates to a whole genome amplification and an immobilization method for its products, belonging to the technical field of genome detection in biomedicine. Background technique [0002] With the deepening of genome research, it will be possible to understand the differences in life at the genetic level, the laws of disease occurrence and development, and the interaction between drugs and living organisms. Although many factors contribute to the occurrence of disease, genetic mutation (SNP, methylation, etc.) is widely recognized as an important intrinsic factor. In basic research, gene mutation sites contribute to the precise positioning of the genome, study the inheritance laws of disease genes, and clone disease-causing genes; Cancer, diabetes, cardiovascular disease, depression, asthma, etc., are affected by many genes and environmental factors. Information on disease-associated genotypes. More importantly, by screening drug-sensiti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 肖鹏峰
Owner SOUTHEAST UNIV
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