阅读说明：本技术 基因文库的构建方法及其应用 (Construction method and application of gene library ) 是由 赵佳 杨林 卢森 蒲丹丹 高健 高雅 陈芳 于 2018-04-09 设计创作，主要内容包括：一种基因文库的构建方法,根据该方法构建得到的基因文库,以及在孕妇无创产前检测领域中的应用。该基因文库的构建方法包括将生物样本和肝素酶共孵育,获得含有生物样本和肝素酶的共孵育样本；从共孵育样本中获取得到DNA,进行高通量建库测序,其中建库过程中利用Taq酶对DNA进行扩增。(A construction method of a gene library, the gene library constructed by the method and application in the field of noninvasive prenatal detection of pregnant women. The construction method of the gene library comprises the steps of incubating a biological sample and heparinase together to obtain an incubated sample containing the biological sample and the heparinase; and obtaining DNA from the co-incubation sample, and performing high-throughput library construction sequencing, wherein Taq enzyme is used for amplifying the DNA in the library construction process.)

A method for constructing a gene library, wherein the gene library is suitable for a biological sample containing heparin, comprises the following steps:

(1) co-incubating the biological sample and heparinase to obtain a co-incubated sample containing the biological sample and the heparinase;

(2) and obtaining DNA from the co-incubation sample, and performing high-throughput library construction sequencing, wherein Taq enzyme is used for amplifying the DNA in the library construction process.

The method according to claim 1, wherein the heparinase in step (1) is heparinase II.

The method according to claim 2, wherein the heparinase is added in an amount of 0.8-2U per 100. mu.L of the biological sample.

The method according to any one of claims 1 to 3, wherein the incubation time with heparinase in step (1) is 20-60 minutes.

The method according to any one of claims 1 to 4, wherein the reaction system for performing the amplification in step (2) comprises: template DNA, Taq enzyme, PCR primers, a buffer solution and ddH2O, wherein the buffer solution comprises dNTP, magnesium ions, Tris-HCl and EDTA-K +.

The method according to any one of claims 1 to 5, wherein the Taq enzyme in step (2) is Invitrogen plantanum Taq enzyme.

The method of any one of claims 1-6, wherein the biological sample is a heparin-containing plasma sample.

The method according to any one of claims 1 to 7, wherein free DNA is extracted from the co-incubated sample, thereby obtaining the resulting DNA.

The method of any one of claims 1 to 7, wherein whole genomic DNA is extracted from the co-incubated sample to obtain the resultant DNA.

A gene library suitable for use in a biological sample containing heparin, the gene library being constructed by the method of any one of claims 1 to 9.

A kit comprising Taq enzyme and heparinase, said kit being suitable for performing an amplification reaction on a biological sample comprising heparin.

The kit according to claim 11, wherein the method according to any one of claims 1 to 9 is used to construct a gene library.

The kit of claim 11, wherein the kit is capable of non-invasive prenatal gene testing of pregnant women.

Use of the gene library of claim 10 or the kit of any one of claims 11-13 in the field of non-invasive prenatal gene testing of pregnant women.

The use according to claim 14, wherein the use is for the diagnosis of a fetal genetic abnormality.

A method for constructing a gene library for noninvasive prenatal testing of pregnant women, which comprises obtaining peripheral blood from the body of a pregnant woman, separating the obtained blood plasma as a biological sample, and constructing the gene library according to the method of claims 1 to 9.

A method of determining a genetic abnormality in a fetus comprising:

a) constructing a gene library of the obtained biological sample by using the method of claims 1 to 9, respectively, to obtain sequence information of a plurality of polynucleotide fragments of the biological sample, respectively;

b) assigning the plurality of polynucleotide fragments of the biological sample to chromosomes based on the sequence information;

c) calculating a coverage depth and a GC content of the chromosome based on the sequence information;

d) calculating a fitted depth of coverage for the chromosome using the GC content of the chromosome and the established relationship between depth of coverage and GC content for the chromosome;

e) comparing the fitted depth of coverage with the depth of coverage of the chromosome, wherein differences between them are used to indicate a fetal genetic abnormality.

The method of claim 17, wherein the relationship in step d) is the following equation:

cr_i，j＝f(GC_i，j)+_i，j，j＝1，2，…，22，X，Y

wherein, f (GC)_i，j) A function representing the relationship between the biological sample i, the depth of coverage of chromosome j and the corresponding GC content,_i，jrepresents the residual error of sample i and chromosome j;

the fitted depth of coverage is calculated according to the following formula:

a system for determining a genetic abnormality in a fetus comprising:

a gene library constructing unit that constructs a gene library of a biological sample obtained by the method according to any one of claims 1 to 9, thereby obtaining sequence information of a plurality of polynucleotide fragments of the biological sample, respectively;

a computer-readable medium of instructions for performing the steps comprising: ,

assigning the plurality of polynucleotide fragments of the biological sample to chromosomes based on the sequence information;

calculating a coverage depth and a GC content of the chromosome based on the sequence information;

calculating a fitted depth of coverage for the chromosome using the GC content of the chromosome and the established relationship between depth of coverage and GC content for the chromosome;

comparing the fitted depth of coverage with the depth of coverage of the chromosome, wherein differences between them are used to indicate a fetal genetic abnormality.

The system of claim 19, wherein the relationship between the depth of coverage and GC content of the chromosome is the following equation:

cr_i，j＝f(GC_i，j)+_i，j，j＝1，2，…，22，X，Y

wherein, f (GC)_i，j) A function representing the relationship between the biological sample i, the depth of coverage of chromosome j and the corresponding GC content,_i，jrepresents the residual error of sample i and chromosome j;

the fitted depth of coverage is calculated according to the following formula: