DNA library construction method for prenatal diagnosis

A DNA library and construction method technology, applied in libraries, chemical libraries, nucleotide libraries, etc., can solve problems such as deviation of final sequencing data, detection errors, loss of cell-free DNA, etc., to reduce cost and time, reduce deviation, The effect of reducing usage

Inactive Publication Date: 2013-10-23
ANNOROAD GENE TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When cell-free DNA is extracted from maternal plasma, the cell-free DNA of the fetus may be

Method used

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  • DNA library construction method for prenatal diagnosis
  • DNA library construction method for prenatal diagnosis
  • DNA library construction method for prenatal diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: The library construction process of the present invention.

[0022] 1. End repair of cell-free DNA in maternal plasma

[0023] DNA extracted from the blood of pregnant women is double-stranded DNA fragments that are either blunt-ended or contain 3' or 5' overhangs. In this step, the overhangs are phosphorylated to blunt ends by T4 DNA polymerase, E. coli DNA polymerase I large fragment (Klenow fragment), and polynucleotide kinase T4 (T4 PNK). 3' to 5' exonuclease activity removes 3' overhangs and polymerase activity fills in 5' overhangs. Finally, the cell-free DNA has blunt ends.

[0024] Material:

[0025] maternal plasma;

[0026] Mixture of dNTPs (10 mM);

[0027] T4 DNA polymerase (3 units / μl);

[0028] Klenow Fragment (5 units / μl);

[0029] T4 PNK (10 units / μl) and PNK buffer;

[0030] Magnetic beads for DNA purification;

[0031] PCR machine.

[0032] program:

[0033] A. Prepare a combination of the following reactions;

[0034]

[0035...

Embodiment 2

[0091] Example 2: Screening of DNA polymerase for PCR in library construction

[0092] In the process of determining the library construction process of the present invention, in order to solve the low yield of the final library, we have screened various DNA polymerases for PCR in various aspects, and finally determined 4 DNA polymerases (including Phusion High- Fidelity PCR Master Mix) for actual experimental comparison. The names and manufacturers of DNA polymerases are as follows:

[0093]

[0094] The specific screening process is as follows:

[0095] 1. End repair of cell-free DNA in maternal plasma

[0096] DNA extracted from the blood of pregnant women are double-stranded DNA fragments that are either blunt-ended or contain 3 or 5' overhangs. This step is to phosphorylate the overhangs to blunt ends by T4 DNA polymerase, E. coli DNA polymerase I large fragment (Klenow fragment), and polynucleotide kinase T4. 3' to 5' exonuclease activity removes 3' overhangs and ...

Embodiment 3

[0189] Example 3: Exploration of PCR amplification conditions in library construction

[0190] After selecting the brand type of DNA polymerase for PCR, we adjusted the system of Platinum® Pfx DNA Polymerase and obtained an amplification system that is more suitable for this experimental process.

[0191] 1. End repair of cell-free DNA in maternal plasma

[0192] DNA extracted from the blood of pregnant women are double-stranded DNA fragments that are either blunt-ended or contain 3 or 5' overhangs. This step is accomplished by phosphorylating the overhangs to blunt ends by T4 DNA polymerase, E. coli DNA polymerase I large fragment (Klenow fragment), and polynucleotide kinase T4. 3' to 5' exonuclease activity removes 3' overhangs and polymerase activity fills in 5' overhangs. Finally, the cell-free DNA has blunt ends.

[0193] Material:

[0194] maternal plasma;

[0195] Mixture of dNTPs (10 mM);

[0196] T4 DNA polymerase (3 units / μl);

[0197] Klenow Fragment (5 units...

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Abstract

The invention provides a DNA library construction method for prenatal diagnosis. According to the DNA library construction method for the prenatal diagnosis, pregnant woman blood plasma is directly used for library construction, and a DNA extraction step is omitted, so that use amount of the pregnant woman blood plasma can be reduced, at the same time, possibility of deviation of fetal DNA content can be reduced, and cost and time for the DNA library construction are greatly reduced. Another aspect of the invention relates to DNA libraries prepared by any of above preparation methods. The third aspect of the invention relates to a DNA sequencing method. The DNA library construction method has good application prospect.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a method for constructing a DNA library for prenatal diagnosis. Background technique [0002] A small number of newborns have congenital genetic diseases or birth defects, which bring huge economic and spiritual burdens to society and families. In 2007, 15.64 million newborns were born in China, of which the birth defect rate exceeded 1%. Birth defects are an important problem affecting the health level and quality of the whole population in my country. The National "Eleventh Five-Year Plan" outlines reproductive health-related technologies as a priority theme for scientific and technological development; the 2006-2020 national medium and long-term plan also lists "birth defect prevention and control" as a key area and priority theme. About 800,000 to 1,200,000 defective babies are born in my country every year, resulting in an...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08C12Q1/68
Inventor 陈重建梁峻彬玄兆伶王占东
Owner ANNOROAD GENE TECH BEIJING
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