Cell chromosome analysis rapid library building method and kit

A chromosome and kit technology, applied in the fields of chemical library, combinatorial chemistry, library creation, etc., can solve the problem of time-consuming and labor-intensive, and achieve the effect of speeding up the test process, stable staining analysis, and low cost

Inactive Publication Date: 2017-05-31
HANGZHOU GENE META MEDICAL DEVICE CO LTD
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0012] Therefore, the entire test process takes 9 hours

Method used

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  • Cell chromosome analysis rapid library building method and kit
  • Cell chromosome analysis rapid library building method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This example is used to illustrate that this method can quickly construct a sequencing library for cell chromosome analysis.

[0050] 1. Mix 20ul human peripheral blood or heel blood with 200ul PBS, centrifuge at 1600g for 10 minutes, remove the supernatant, and hang the cells in 50ul PBS.

[0051] 2. Add 50ul of 20mg / ml proteinase K, inactivate at 60°C for 20 minutes, and at 95°C for 5 minutes.

[0052] 3. Add 10 units / ul DNase I and DNase II and DNA end filling reagent 2 units / ul end filling enzyme (T4 DNA polymerase or Klenowexo-), 490uM dNTPs, 55mM Tris Buffer composition, reaction conditions are 37°C for 20 minutes, 75°C for 5 minutes for inactivation.

[0053] 4. Add DNA ligation reagent directly into the test tube: 100 units / ul of DNA ligase, 3.5mM of ATP, 55mM of Tris buffer, 30mM of dithiothreitol and DNA linker. The reaction condition is 20°C for 20 minutes. After the reaction is completed, it can be directly purified by silica gel column or magnetic particl...

Embodiment 2

[0056] This example is used to illustrate that this method can quickly construct a chromosome analysis and sequencing library of fetal amniotic fluid exfoliated cells.

[0057] 1. Centrifuge 2-5ml amniotic fluid at 1600g for 10 minutes, remove the supernatant, and hang the cells in 50ul PBS.

[0058] 2. Add 50ul of 20mg / ml proteinase K, inactivate at 60°C for 20 minutes, and at 95°C for 5 minutes.

[0059] 3. Add 10 units / ul DNase I and DNase II and DNA end filling reagent 2 units / ul end filling enzyme (T4 DNA polymerase or Klenowexo-), 490uM dNTPs, 55mM Tris Buffer composition, reaction conditions are 37°C for 20 minutes, 75°C for 5 minutes for inactivation.

[0060] 4. Add DNA ligation reagent directly into the test tube: 100 units / ul of DNA ligase, 3.5mM of ATP, 55mM of Tris buffer, 30mM of dithiothreitol and DNA linker. The reaction condition is 20°C for 20 minutes. After the reaction is completed, it can be directly purified by silica gel column or magnetic particles, a...

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Abstract

The invention discloses a cell chromosome analysis rapid library building method. The method comprises the following steps: (1) directly adding proteinase K into cells for cracking the cells; (2) directly adding DNA cracking enzyme and a DNA end filling reagent into the reaction system of the step 1; and (3) directly adding a DNA ligation reagent into the reaction system of the step 2, so that the target to-be-detected DNA is in joint connection with the DNA. The sequencing library provided by the invention can be used for detection of a high-throughput sequencing instrument, the single-tube test can be realized, the genome DNA extraction, the PCR amplification and the purification among the steps are not needed, and thus the chromosome sequencing detection based on micro cells is enabled to be rapid, simple and convenient. The invention further discloses a cell chromosome analysis rapid library building kit.

Description

technical field [0001] The invention belongs to the field of cell chromosome detection, and in particular relates to a method for constructing a DNA sequencing library and a kit for detecting chromosome structure variation. Background technique [0002] Every human cell has 23 pairs of chromosomes. In the genetic and natural process, the structure of some chromosomes has changed, resulting in various birth defects and unexplained miscarriages. Common chromosomal structural variations include the following categories: [0003] 1. Deletion: the deletion of a certain segment of a chromosome. For example, meowing syndrome is a genetic disease caused by a partial deletion of chromosome 5 in humans. It is named because the sick child cries softly and has a high pitch, which resembles a cat meowing. [0004] 2. Duplication: A certain segment of chromosome is added. The rod eye phenomenon of Drosophila is caused by the partial duplication on the X chromosome. [0005] 3. Inversion...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B50/06
CPCC12Q1/6869C40B50/06
Inventor 祝云英
Owner HANGZHOU GENE META MEDICAL DEVICE CO LTD
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