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DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing

A DNA array and targeted capture technology, applied in the field of molecular biology, can solve the problems of high price, high cost, and low resolution, and achieve the effect of rapid screening and low cost

Inactive Publication Date: 2015-09-09
李卫东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescence in situ hybridization (FISH) is based on known specific DNA sequences, using fluorescently labeled specific oligonucleotide fragments as probes to hybridize with the DNA molecules of the detected chromosomes, If the target DNA on the detected chromosome is homologous and complementary to the probe used, a hybrid between the target DNA and the probe can be formed, and the hybridization signal is detected by a fluorescent detection system; these have high requirements for technical operation and interpretation Chromosomal fluorescence in situ hybridization probes are expensive and only target a single site; and chromosome technology is only suitable for detecting cells in division phase, with low resolution, and accurate interpretation of results requires long-term professional training
Chromosomal fluorescence in situ hybridization can detect mitotic cells and interphase cells, but it can only detect one abnormality with one known probe each time, with a high rate of missed detection and strict technical requirements. higher cost

Method used

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  • DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing
  • DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing
  • DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Design of a DNA array for high-throughput targeted capture sequencing of leukemia chromosomal aberrations

[0015] Agilent SureSelect DNA Design Tool was used to design probes for 18 genes (see Table 2) involved in chromosomal aberrations. The parameters are set as follows:

[0016] Species: H.sapiens, hg19, GRCH37, February2009; Databases: RefSeq, Ensembl, CCDS, Gencode, VEGA, SNP,

[0017] CytoBand; Region: Entire Transcribed Region; Region Extension: 10 bases from 3’end and 10 bases from 5’end; Tiling density: 2×; Masking: Least Stringent; Boosting: Balanced.

[0018] Table 2 Gene fragments captured by probes

[0019] serial number Gene chromosome start site stop site Region size (bp) 1 ABL1 (ABL) 9 133589258 133763072 173815 2 ETV6(TEL) 12 11802778 12048335 24558 3 IGHD (IGH) 14 106303089 106312020 8932 4 KAT6A(MOZ) 8 41786987 41909515 122529 5 KMT2A (MLL) 11 118307195 118397549 90355 ...

Embodiment 2

[0020] Embodiment 2 uses the present invention to detect clinical samples

[0021] 1. Extraction of bone marrow or peripheral blood samples to extract genomic DNA.

[0022] 2. Preparation before hybridization

[0023] 2.1 Genomic DNA is broken into small fragments of several hundred bases to build a library.

[0024] 2.2 Use Agencourt AMPure XP beads to purify the sample.

[0025] 2.3 Add base "A" to the 3' end of the DNA fragment.

[0026] 2.4 Use Agencourt AMPure XP beads to purify the sample.

[0027] 2.5 Add double-ended adapters.

[0028] 2.6 Use Agencourt AMPure XP beads to purify the sample.

[0029] 2.7 Amplify the library with adapters.

[0030] 2.8 Use Agencourt AMPure XP beads to purify the sample.

[0031] 2.9 Assess library quality.

[0032] 3. Hybridize and capture the product with the established library.

[0033] 4. The captured DNA fragments are denatured into single strands and combined with the adapter primers on the sequencing channel to form a bri...

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Abstract

The invention discloses a DNA array for leukemia chromosome aberration high-throughput targeted capture detection. In the DNA array, the contained probe is capable of specific targeted capture of 18 genes involved in 37 chromosome aberrations. A sample and the DNA array are hybridized, chromosome aberration involved gene sequences are captured targetedly, then the captured sequences are subjected to sequencing, the sequencing result is analyzed to screen out the existent chromosome aberration type. The probe contained in the DNA array can specifically bind the 18 genes involved in 37 chromosome aberrations so as to realize rapid screening of leukemia related chromosome translocation. By chip inspection, a plurality of samples can be detected at a time, and the cost is low. For the to-be-detected 37 sites, the accuracy is close to 100%. And cytogenetics professionals are not needed for interpretation of the result.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a DNA target capture array for high-throughput sequencing of leukemia chromosome aberrations. Background technique [0002] With the development of molecular cytogenetics technology, it has been confirmed that the specific changes of certain chromosomes are closely related to the occurrence, development and prognosis of leukemia. The detection of chromosomal abnormalities provides an important basis for the clinical diagnosis, staging, treatment, prognosis of leukemia and the treatment of minimal residual disease. [0003] At present, high-resolution banding technology and fluorescence in situ hybridization (FISH) are used to detect chromosomal aberrations in leukemia at home and abroad. Chromosome banding technology refers to the use of special processing procedures to make certain parts of chromosomes Shows different shades of chromosome banding. These b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李卫东杨付花
Owner 李卫东
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