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A method for detecting chromosomal abnormality and recombination site dna sequence
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A DNA sequence and recombination site technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as sequencing chromosomal recombination at low cost, and achieve the effect of overcoming the short read length of NGS and reducing the cost of sequencing
Active Publication Date: 2021-03-09
SHANGHAI TOLO BIOTECH CO LTD
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[0006] Aiming at the problem that existing technical solutions cannot quickly and cheaply sequence the DNA sequence of chromosomal recombination (translocation, inversion, etc.) site, the present invention provides a new method for detecting the DNA sequence of recombination site
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Embodiment 1
[0045] (1) Use classic karyotype analysis, FISH, BioNano and other techniques to determine the status of chromosomal abnormalities (including on which chromosome, what kind of abnormality, and approximate location), such as figure 1 As shown, the chromosomal abnormality is a chromosomal inversion.
[0046] Locate the approximate region of the chromosomal inversion site (such as the region of 1-5M).
[0047] (2) Design the sorted sgRNA sequence for this region, and prepare sgRNA in vitro.
[0049] (4) Inactivate the Cas9 enzyme, and use DNA gap translation technology to incorporate base analogs (such as Biotin-modified dGTP) at the gap.
[0050] (5) DNA is randomly interrupted; the double strand is unwound into single-stranded DNA, and the single-stranded DNA is purified using the characteristics of base ana...
Embodiment 2
[0055](1) Use classic karyotype analysis, FISH, BioNano and other techniques to determine the status of chromosomal abnormalities (including on which chromosome, what kind of abnormality, and approximate location), such as figure 2 As shown, the chromosomal abnormality is an interchromosomal translocation.
[0056] Locate the approximate region of the chromosomal inversion site (such as the region of 1-5M).
[0057] (2) Design and sort the sgRNA sequence for the c1 region of the first chromosome, and prepare the sgRNA in vitro.
[0058] (3) Extract chromosomal DNA; according to CRISPR technology, use Cas9 with single enzymecutting activity (such as D10A or H840A) to cut the chromosome in vitro.
[0059] (4) Inactivate the Cas9 enzyme, and use DNA gap translation technology to incorporate base analogs (such as Biotin-modified dGTP) at the gap.
[0060] (5) DNA is randomly interrupted; the double strand is unwound into single-stranded DNA, and the single-stranded DNA is puri...
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Abstract
The present invention provides a new method for detecting chromosomal abnormalities and DNA sequences of recombination sites. Through relatively mature existing technologies, the approximate region of chromosomal recombination can be analyzed under low-cost conditions, and Cas9 multi-point single-strand cutting technology in vitro , effectively overcome the shortcomings of NGS such as short read length, so that the NGS platform can be directly used for sequencing, which greatly reduces the cost of sequencing, not only can determine information such as chromosomal inversions and translocations, but also accurately determine the DNA sequence of the target region . Compared with traditional chromosomal abnormality detection methods, this method can obtain more DNA sequence information.
Description
technical field [0001] The invention relates to a method for detecting chromosomal abnormality, in particular to a method capable of quickly and low-cost determination of chromosomal abnormality, DNA sequence of abnormal chromosomal recombination region and finding recombination site. Background technique [0002] Birth defects have seriously affected the quality of the population and brought huge losses to the country, society and families. Chromosomal structural and quantitative abnormalities are the main causes of birth defects. Chromosomal structural abnormalities include chromosomal translocations and chromosomal inversions. [0003] A chromosomal translocation is a change in the position of a segment of a chromosome. When a translocation occurs within a single chromosome, it is called a translocation or intrachromosomal translocation, while when it occurs between two homologous or nonhomologous chromosomes, it is called an interchromosomal translocation. [0004] Chr...
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