Method for separating and enriching free fetus DNA from maternal blood plasma

A technology for separation, enrichment, and plasma, applied in the field of separation and enrichment of cell-free fetal DNA, can solve the problems of low cffDNA content, low recovery rate, loss of samples, etc., and achieves the effect of removing DNA interference, good application prospects, and simple method.

Active Publication Date: 2015-08-26
GUANGZHOU KANGRUI BIOLOGICAL PHARMA TECH CO LTD
View PDF0 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the content of cffDNA in the peripheral blood of pregnant women is very small, only about 19% of the total free DNA in plasma, and it is mixed with a large amount of free DNA in maternal plasma, which has caused great difficulties in the detection and isolation of cffDNA. Efficient enrichment of cffDNA in blood has become the focus of research
[0004] At present, there are electrophoretic separation and

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for separating and enriching free fetus DNA from maternal blood plasma
  • Method for separating and enriching free fetus DNA from maternal blood plasma
  • Method for separating and enriching free fetus DNA from maternal blood plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Using the method of the present invention to enrich and purify fetal DNA from plasma of pregnant women

[0084] 1. Experimental method

[0085] 4 pregnant women with a gestational age of 12-16 weeks, samples 1-3 are male fetuses, and sample 4 is female fetuses. Take 5ml of venous blood from pregnant women, and centrifuge the freshly obtained BD blood collection tube (EDTA whole blood) at 4°C, 1900×g for 10 minutes; draw the supernatant into a 15ml conical bottom centrifuge tube, and then centrifuge at 4°C, 16000×g After 10 minutes, carefully transfer the supernatant to a new preservation tube, and use the following methods for cell-free fetal DNA extraction:

[0086] 1. Add 100 μl proteinase K to a 15ml centrifuge tube;

[0087] 2. Add 1ml of maternal plasma to the centrifuge tube;

[0088] 3. Add 0.9ml of binding buffer A, vortex for 15-30s, and mix well;

[0089] 4. Incubate at 60°C for 30 minutes;

[0090] 5. Add the lysate to the silica column twice, ...

Embodiment 2

[0125] Example 2 The method of the present invention is used to extract the male-specific SRY gene in plasma

[0126] In this example, the effectiveness of the method of the present invention is verified by adding SRY gene fragments to blank plasma. The SRY gene fragment of the present invention has a length of 194bp and belongs to free fetal DNA.

[0127] 1. Experimental method

[0128] There are Y chromosome-specific sequences in male fetal DNA. Currently, researchers usually use fluorescent quantitative PCR technology to detect specific genes on the Y chromosome, such as SRY gene, DYS14, etc., so as to quantify male free fetal DNA.

[0129] The SRY gene is a male-specific gene. Usually the free DNA content in human plasma is about 1–100ng / ml. Two parts of 600 μl of plasma come from a 75-year-old female, and the nucleotide sequence such as SEQ ID NO.1 is added according to the ratio of the following table 1

[0130](aattgcagtttgcttcccgcagatcccgcttcggtactctgcagcgaagtgcaac...

Embodiment 3

[0138] Embodiment 3 The present invention selects the screening experiment of eluent

[0139] 1. Experimental method

[0140] The operation steps are as follows, and step 7 is processed according to three methods:

[0141] 1. 5ul 1kb plus DNA ladder plus 1ml H 2 In the centrifuge tube of O;

[0142] 2. Add 0.9ml of binding buffer A, vortex for 15-30s, and mix well;

[0143] 4. Incubate at 60°C for 30 minutes;

[0144] 5. Add the lysate to the silica column twice, 1ml each time;

[0145] 6. Centrifuge at 13000rpm for 1min, discard the collection tube, and replace with a new collection tube;

[0146] 7. Treatment 1: Add 750 μl of washing buffer B, centrifuge at 13,000 rpm for 1 min, discard the collection tube, and replace it with a new collection tube, the nucleic acid is still bound to the membrane, and the residual pollutants such as protein and divalent cations are effectively washed away;

[0147] Or treatment 2: Add 750 μl of washing buffer C1 (treatment 2 is selecti...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention discloses a method for separating and enriching small DNA fragments from a DNA-containing sample. The method comprises: centrifugating a sample to prepare a cell-free sample, lysing, binding to a silica column, washing, carrying out partial elution, re-binding, washing, and carrying out elution. With the method of the present invention, the low content free fetus DNA in the blood plasma can be effectively enriched, the method is simple, practical and effective, and is suitable for simultaneous treatment of multiple samples, harmful substances such as phenol-chloroform and the like are not required, the obtained small DNA fragment has the high purity and can be used directly in downstream PCR, real time fluorescence quantitative PCR, sequencing and the like, and the good application prospects are provided.

Description

technical field [0001] The invention relates to a method for separating and enriching free fetal DNA from maternal plasma, belonging to the field of biotechnology. Background technique [0002] In my country, there are as many as 800,000 to 1.2 million newborns with birth defects every year. Prenatal screening and targeting are the most effective countermeasures. However, the current conventional targeting methods are all innovative methods, which are harmful to fetuses and pregnant women. Therefore, the development of new non-invasive prenatal diagnosis has important clinical significance. In 1997, Lo et al. found that there was a small amount of free fetal DNA (cffDNA) in the plasma of pregnant women, which made it possible to develop maternal plasma for safe non-invasive prenatal diagnosis (NIPD). [0003] However, the content of cffDNA in the peripheral blood of pregnant women is very small, only about 19% of the total free DNA in plasma, and it is mixed with a large amo...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/10
Inventor 罗红蓉
Owner GUANGZHOU KANGRUI BIOLOGICAL PHARMA TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap