DNA library construction method in fetal chromosome aneuploid detection

Abstract

The invention belongs to the technical field of fetal chromosome aneuploid detection, and particularly relates to a DNA library construction method in fetal chromosome aneuploid detection, and the method comprises the following steps: S1, carrying out terminal repair and 3'A addition on DNA; s2, connecting a sequencing joint and a tag; s3, purifying the DNA; s4, carrying out amplification on the purified DNA; according to the method, a PCR reinforcing agent is added in the amplification process to improve the amplification efficiency, a fragment screening step is added in library construction, fragment purification is carried out according to the concentration of magnetic beads, the concentration of fetal DNA can be effectively increased, the content of fetal DNA in peripheral blood is increased, impurity residues are reduced, and the detection sensitivity is improved. Moreover, the enzyme activity is not influenced, and the uniformity and quality of sequencing data are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of fetal chromosome aneuploidy detection, and particularly relates to a method for constructing a DNA library in fetal chromosome aneuploidy detection. Background Art

[0002] There are 23 pairs, a total of 46 chromosomes in a normal human body. Except for sex chromosomes, each type of chromosome has 2 copies. If a chromosomal copy number deletion / duplication is found, chromosomal aneuploidy will occur, which is one of the common causes of human genetic diseases. The incidence of chromosomal abnormalities in newborns is 1 / 160. Among them, trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13) are the three most common autosomal aneuploidy diseases.

[0003] Since there is free fetal DNA in maternal peripheral blood, fetal DNA fragments are generally shorter than those of the mother, and fetal chromosomal abnormalities will cause a slight change in the DNA content in the mother. This change can be detected through deep sequencing and bioinformatics analysis. Circulating free fetal DNA accounts for about 3-13% of the mother. For most methods of constructing DNA libraries in fetal chromosome aneuploidy detection, in the case of less fetal free DNA content in twin pregnancies, false negative and false positive results may occur. Existing DNA library construction methods cannot detect other types of fetal abnormalities (such as structural abnormalities or certain genetic diseases), and most of these methods are manual construction methods, with a large influence of human factors, which will have a certain impact on the sequencing results. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a method for constructing a DNA library in fetal chromosome aneuploidy detection, aiming to increase the content of fetal DNA in peripheral blood, reduce impurity residues, and not affect enzyme activity, thereby improving the uniformity and quality of sequencing data.

[0005] The present invention solves its technical problems by adopting the following technical solutions:

[0006] The present invention aims to provide a method for constructing a DNA library in fetal chromosome aneuploidy detection, comprising the following steps:

[0007] S1. Perform end repair and 3'-end adenylation on DNA;

[0008] S2. Connect sequencing adapters and tags;

[0009] S3. Purify DNA;

[0010] S4. Amplify the purified DNA;

[0011] S5. Purify the amplified product by fragment screening.

[0012] Furthermore, in 1 - 5 ng of free DNA, use 2 μL of end repair enzyme and 10 μL of buffer to blunt-end and add a poly(A) tail to the free DNA in a PCR instrument.

[0013] Furthermore, the reaction program in the PCR instrument is: 4°C for 1 min; 32°C for 30 min; 65°C for 20 min; 4°C, Hold.

[0014] Furthermore, the method for ligating sequencing adapters and tags includes: adding 14 μL of T4 DNA ligase buffer, 3 μL of T4 DNA ligase, 5 μL of Adaptor, 5 μL of Index, and 3 μL of NF water to the reactant after the S1 reaction, and the reaction program in the PCR instrument is 23°C for 20 min.

[0015] Furthermore, in S3, use one of the following magnetic beads for DNA purification: Ampure XP magnetic beads, KAPA Pure magnetic beads, VAHTS DNA Clean magnetic beads, ProNex magnetic beads, TIANSeq DNA fragment sorting magnetic beads, MagicPure Size selection DNA magnetic beads.

[0016] Furthermore, the method for purifying DNA in S3 includes:

[0017] 1) Pre-equilibrate the magnetic beads for 20 - 40 min;

[0018] 2) Add 1x volume of magnetic beads to the ligation product, mix well by pipetting up and down, and let it stand at room temperature for 3 - 8 minutes;

[0019] 3) Place the PCR tube on a magnetic rack and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the inner wall of the tube close to the magnetic rack, and discard the supernatant;

[0020] 4) Keep the PCR tube on the magnetic rack, add 180 - 220 μL of freshly prepared 80% ethanol to the tube, let it stand at room temperature for 20 - 40 seconds, and discard the supernatant;

[0021] 5) Repeat step 4);

[0022] 6) Keep the PCR tube on the magnetic rack and let it dry at room temperature until the magnetic beads crack;

[0023] 7) Remove the PCR tube from the magnetic stand, add no less than 20 μL of NF Waer or TE to elute the DNA, pipette up and down to mix well, or vortex to mix well, and let it stand at room temperature for 1 - 3 minutes;

[0024] 8) Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic stand.

[0025] Furthermore, in S4, the PCR enhancer used for amplifying the purified DNA is bovine serum albumin and 1 - butyl - 3 - methylimidazolium bromide.

[0026] Furthermore, the concentration of bovine serum albumin is 1 - 10 mg / mL, preferably 5 mg / mL, and the concentration of 1 - butyl - 3 - methylimidazolium bromide is 1.5 mol / L.

[0027] Furthermore, the PCR amplification reaction program is as follows:

[0028] 98 °C, 3 min;

[0029] 98 °C, 30 s; 60 °C, 30 s; 72 °C, 30 s; set the number of cycles to 15;

[0030] 72 °C, 5 min;

[0031] 4 °C, Hold.

[0032] Furthermore, the method for fragment screening in S5 includes:

[0033] 1) Equilibrate the magnetic beads in advance for 20 - 40 min, and add 0.8x volume of magnetic beads to the amplified product;

[0034] 2) Pipette up and down to mix well, or vortex to mix well, and let it stand at room temperature for 3 - 8 minutes;

[0035] 3) Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic stand;

[0036] 4) Keep the PCR tube on the magnetic stand, transfer the supernatant to another clean centrifuge tube, and discard the magnetic beads;

[0037] 5) Add 0.2x volume of magnetic beads to the supernatant, pipette up and down to mix well, and let it stand at room temperature for 3 - 8 minutes;

[0038] 6) Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic stand, and discard the supernatant;

[0039] 7) Keep the PCR tube on the magnetic stand, add 180 - 220 μL of freshly prepared 80% ethanol to the tube, let it stand at room temperature for 20 - 40 seconds, and discard the supernatant;

[0040] 8) Repeat step 7);

[0041] 9) Keep the PCR tube on the magnetic stand and air-dry it at room temperature until the magnetic beads crack;

[0042] 10) Remove the PCR tube from the magnetic stand, add no less than 20 μL of Nuclease-free Water or TE to elute the DNA, pipette and mix well, or vortex mix, and let it stand at room temperature for 1 - 3 minutes;

[0043] 11) Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic stand;

[0044] 12) Transfer the eluate to a clean centrifuge tube, measure the concentration, and store it at -15 to -25 °C.

[0045] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0046] 1. In the amplification process of the present invention, a PCR enhancer is added to improve the amplification efficiency, and a new fragment screening step is added in the library construction. The fragments are purified by the concentration of magnetic beads, which can effectively increase the concentration of fetal DNA and screen for DNA fragments of about 140 bp.

[0047] 2. The PCR enhancer combination of the present invention is bovine serum albumin and 1-butyl-3-methylimidazolium bromide, which can further improve the efficiency of the PCR amplification reaction. Bovine serum albumin can competitively aggregate at the oil-water interface, thereby reducing the interfacial adsorption of DNA polymerase; bovine serum albumin can improve the amplification efficiency of the PCR reaction; 1-butyl-3-methylimidazolium bromide can improve the stability of the DNA structure and increase the PCR amplification efficiency.

[0048] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the content of the specification. And in order to make the above content, its purpose, features and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention are specifically given. Brief Description of the Drawings

[0049] Figure 1 It is the quality inspection chart of the 2100 fragments in Comparative Example 1 of the present invention.

[0050] Figure 2 It is the quality inspection chart of the 2100 fragments in Comparative Example 2 of the present invention.

[0051] Figure 3 It is the quality inspection chart of the 2100 fragments in Comparative Example 3 of the present invention.

[0052] Figure 4 This is the quality inspection diagram of the 2100 fragment in Test Example 1 of the present invention. Detailed implementation manners

[0053] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0054] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0055] The method for constructing a DNA library in fetal chromosomal aneuploidy detection mainly includes the following steps: 1. Obtain free DNA from pregnant women's peripheral blood; 2. Perform end repair and 3'-adenylation on the DNA; 3. Ligate sequencing adapters and special tags; 4. Purify the DNA; 5. Amplify the purified DNA (with enhancer); 6. Purify the amplified product by fragment screening.

[0056] Example 1

[0057] The detailed process of the method for constructing a DNA library in fetal chromosomal aneuploidy detection is as follows:

[0058] 1 End repair and addition of A tail

[0059] 1.1 Use end repair enzymes and buffers to perform blunt-end ligation and addition of A tail to the free DNA. Add the following reagents (ComWin Biotech or other manufacturers) to a 200 μL PCR tube:

[0060]

[0061] 1.2 Flick to mix evenly, place it in a set PCR instrument, and start the reaction. The program is as follows:

[0062]

[0063]

[0064] 2 Ligation of adapters and tags

[0065] 2.1 Immediately perform adapter ligation reaction after the reaction ends. Add the following reagents to the previous PCR tube:

[0066]

[0067] 2.2 Vortex to mix evenly, centrifuge briefly and place on ice, then place it in a set PCR instrument and start the reaction. The program is as follows:

[0068]

[0069] 3. Purify the DNA after ligation

[0070] 3.1 Use any of the following Ampure XP magnetic beads (Beckman), KAPA Pure Beads (KAPA), VAHTS DNA Clean Beads (vazyme), ProNex Beads (for size selection) (Promega), TIANSeq DNA fragment sorting magnetic beads (TIAGEN), and MagicPure Size selection DNA Beads (TRANS) for purification. Equilibrate the magnetic beads half an hour in advance.

[0071] 3.2 Add 1x volume of magnetic beads to the ligation product, pipette up and down to mix well, and let stand at room temperature for 5 minutes.

[0072] 3.3 Place the PCR tube on the magnetic stand and let stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand. Discard the supernatant.

[0073] 3.4 Keep the PCR tube on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to the tube, do not pipette up and down the magnetic beads, and let stand at room temperature for 30 seconds. Discard the supernatant.

[0074] 3.5 Repeat step 3.4.

[0075] 3.6 Keep the PCR tube on the magnetic stand and air-dry at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0076] 3.7 Remove the PCR tube from the magnetic stand, add no less than 20 μL of NF Water or TE to elute the DNA. Pipette up and down to mix well, or vortex to mix well and let stand at room temperature for 2 minutes.

[0077] 3.8 Place the PCR tube on the magnetic stand and let stand at room temperature until the solution becomes clear (about 2 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0078] 3.9 Transfer the eluate to a clean PCR tube for the next step.

[0079] 4. PCR amplification

[0080] 4.1 Prepare the following PCR reaction system:

[0081]

[0082] In addition to containing conventional reagents such as polymerase, buffer, dNTPs, MgCl2, etc., the PCR mix also contains a combination of PCR enhancers of bovine serum albumin and 1-butyl-3-methylimidazolium bromide, where the concentration of bovine serum albumin is 5 mg / mL and the concentration of 1-butyl-3-methylimidazolium bromide is 1.5 mol / L.

[0083] 4.2 After mixing well by shaking, place it in a set PCR instrument to start the reaction. The program is as follows:

[0084]

[0085] 5. Fragment screening

[0086] 5.1 Equilibrate the magnetic beads in advance for 30 min, and add 0.8x volume of magnetic beads to the amplified product.

[0087] 5.2 Mix well by pipetting up and down, or by vortexing, and let it stand at room temperature for 5 minutes.

[0088] 5.3 Place the PCR tube on the magnetic rack and let it stand at room temperature until the solution becomes clear (about 5 minutes) so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic rack.

[0089] 5.4 Keep the PCR tube on the magnetic rack, transfer the supernatant to another clean 1.5 ml centrifuge tube, and discard the magnetic beads.

[0090] 5.5 Add 0.2x volume of magnetic beads to the supernatant, mix well by pipetting up and down, and let it stand at room temperature for 5 minutes.

[0091] 5.6 Place the PCR tube on the magnetic rack and let it stand at room temperature until the solution becomes clear (about 5 minutes) so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic rack. Discard the supernatant.

[0092] 5.7 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to the tube, do not pipette up and down the magnetic beads, and let it stand at room temperature for 30 seconds. Discard the supernatant.

[0093] 5.8 Repeat 5.7.

[0094] 5.9 Keep the PCR tube on the magnetic rack and let it dry at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0095] 5.10 Remove the PCR tube from the magnetic rack, add no less than 20 μL of Nuclease-free Water or TE to elute the DNA. Mix well by pipetting up and down, or by vortexing, and let it stand at room temperature for 2 minutes.

[0096] 5.11 Place the PCR tube on the magnetic rack and let it stand at room temperature until the solution becomes clear (about 2 minutes) so that the magnetic beads are fully adsorbed on the tube wall close to the magnetic rack.

[0097] 5.12 Transfer the eluate to a clean centrifuge tube. After measuring the concentration, store it at -20°C.

[0098] Example 2

[0099] The detailed process of the DNA library construction method in fetal chromosomal aneuploidy detection is as follows:

[0100] 1 End repair and A-tailing

[0101] 1.1 Use end repair enzymes and buffers to blunt-end and add A-tail to the cell-free DNA. Add the following reagents (ComWin Biotech or other manufacturers) to a 200 μL PCR tube:

[0102]

[0103] 1.2 Flick to mix well, place it in a set PCR instrument, and start the reaction. The program is as follows:

[0104]

[0105] 2 Ligation of adapters and tags

[0106] 2.1 Immediately after the reaction, perform the adapter ligation reaction. Add the following reagents to the previous PCR tube:

[0107]

[0108] 2.2 Vortex to mix well, centrifuge briefly and place on ice, then put it in a set PCR instrument and start the reaction. The program is as follows:

[0109]

[0110] 3. Purify the DNA after ligation

[0111] 3.1 Purify using any of the following Ampure XP magnetic beads (Beckman), KAPA Pure Beads (KAPA), VAHTS DNA Clean Beads (vazyme), ProNex Beads (for size selection) (Promega), TIANSeq DNA fragment sorting magnetic beads (TIAGEN), and MagicPure Size selection DNA Beads (TRANS). Equilibrate the magnetic beads for 20 min in advance.

[0112] 3.2 Add 1x volume of magnetic beads to the ligation product, pipette to mix well, and let stand at room temperature for 3 minutes.

[0113] 3.3 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand. Discard the supernatant.

[0114] 3.4 Keep the PCR tube on the magnetic stand and add 180 μL of freshly prepared 80% ethanol to the tube. Do not pipette the magnetic beads and let it stand at room temperature for 20 seconds. Discard the supernatant.

[0115] 3.5 Repeat step 3.4.

[0116] 3.6 Keep the PCR tube on the magnetic stand and air-dry it at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0117] 3.7 Remove the PCR tube from the magnetic stand and add no less than 20 μL of NF Water or TE to elute the DNA. Mix well by pipetting up and down or vortexing and let it stand at room temperature for 1 minute.

[0118] 3.8 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 2 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0119] 3.9 Transfer the eluate to a clean PCR tube for the next step.

[0120] 4. PCR Amplification

[0121] 4.1 Prepare the PCR reaction system as follows:

[0122]

[0123] In addition to containing conventional reagents such as polymerase, buffer, dNTPs, MgCl2, etc., the PCR mix also contains a combination of PCR enhancers of bovine serum albumin and 1-butyl-3-methylimidazolium bromide, where the concentration of bovine serum albumin is 1 mg / mL and the concentration of 1-butyl-3-methylimidazolium bromide is 1.5 mol / L.

[0124] 4.2 After mixing by shaking, place it in the set PCR instrument to start the reaction. The program is as follows:

[0125]

[0126]

[0127] 5. Fragment Screening

[0128] 5.1 Pre-equilibrate the magnetic beads for 20 min and add 0.8x volume of magnetic beads to the amplified product.

[0129] 5.2 Mix well by pipetting up and down or vortexing and let it stand at room temperature for 3 minutes.

[0130] 5.3 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0131] 5.4 Keep the PCR tube on the magnetic stand and transfer the supernatant to another clean 1.5 ml centrifuge tube, discarding the magnetic beads.

[0132] 5.5 Add magnetic beads with a volume of 0.2x to the supernatant, mix well by pipetting up and down, and let it stand at room temperature for 3 minutes.

[0133] 5.6 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand. Discard the supernatant.

[0134] 5.7 Keep the PCR tube on the magnetic stand and add 180 μL of freshly prepared 80% ethanol to the tube. Do not pipette up and down the magnetic beads and let it stand at room temperature for 20 seconds. Discard the supernatant.

[0135] 5.8 Repeat step 5.7.

[0136] 5.9 Keep the PCR tube on the magnetic stand and let it air dry at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0137] 5.10 Remove the PCR tube from the magnetic stand and add no less than 20 μL of Nuclease - free Water or TE to elute the DNA. Mix well by pipetting up and down or vortexing, and let it stand at room temperature for 1 minute.

[0138] 5.11 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 2 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0139] 5.12 Transfer the eluate to a clean centrifuge tube, measure the concentration, and store it at - 15°C.

[0140] Example 3

[0141] The detailed process of the DNA library construction method in fetal chromosomal aneuploidy detection is as follows:

[0142] 1 End repair and A - tail addition

[0143] 1.1 Use end repair enzymes and buffers to blunt - end and add an A - tail to the cell - free DNA. Add the following reagents (ComWin Biotech or other manufacturers) to a 200 μL PCR tube:

[0144]

[0145] 1.2 Mix gently and place it in a pre - set PCR instrument to start the reaction. The program is as follows:

[0146]

[0147] 2 Ligation Adapter and Label

[0148] 2.1 Immediately perform the ligation reaction after the reaction ends. Add the following reagents to the previous PCR tube:

[0149]

[0150] 2.2 Vortex to mix well, briefly centrifuge and place on ice, then put it into the set PCR instrument to start the reaction. The program is as follows:

[0151]

[0152] 3. Purify DNA after ligation

[0153] 3.1 Use any of the following Ampure XP magnetic beads (Beckman), KAPA Pure Beads (KAPA), VAHTS DNAClean Beads (vazyme), ProNex Beads (for size selection) (Promega), TIANSeq DNA fragment sorting magnetic beads (TIAGEN), and MagicPure Size selection DNA Beads (TRANS) for purification. Equilibrate the magnetic beads in advance for 40 min.

[0154] 3.2 Add 1x volume of magnetic beads to the ligation product, pipette up and down to mix well, and let it stand at room temperature for 8 minutes.

[0155] 3.3 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb on the tube wall close to the magnetic stand. Discard the supernatant.

[0156] 3.4 Keep the PCR tube on the magnetic stand, add 220 μL of freshly prepared 80% ethanol to the tube, do not pipette up and down the magnetic beads, and let it stand at room temperature for 40 seconds. Discard the supernatant.

[0157] 3.5 Repeat step 3.4.

[0158] 3.6 Keep the PCR tube on the magnetic stand and air-dry it at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0159] 3.7 Remove the PCR tube from the magnetic stand, add no less than 20 μL of NF Waer or TE to elute the DNA. Pipette up and down to mix well, or vortex to mix well and let it stand at room temperature for 3 minutes.

[0160] 3.8 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 2 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0161] 3.9 Transfer the eluate to a clean PCR tube for the next step.

[0162] 4. PCR Amplification

[0163] 4.1 Prepare the PCR reaction system as follows:

[0164]

[0165] In addition to conventional reagents such as polymerase, buffer, dNTPs, MgCl2, etc., the PCR mix also contains a combination of PCR enhancers, bovine serum albumin and 1-butyl-3-methylimidazolium bromide, with the concentration of bovine serum albumin being 10 mg / mL and the concentration of 1-butyl-3-methylimidazolium bromide being 1.5 mol / L.

[0166] 4.2 After mixing by vortexing, place it in a pre-set PCR instrument to start the reaction. The program is as follows:

[0167]

[0168] 5. Fragment Screening

[0169] 5.1 Equilibrate the magnetic beads for 40 min in advance and add 0.8x volume of magnetic beads to the amplified product.

[0170] 5.2 Mix well by pipetting up and down or by vortexing, and let it stand at room temperature for 8 minutes.

[0171] 5.3 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0172] 5.4 Keep the PCR tube on the magnetic stand and transfer the supernatant to another clean 1.5 mL centrifuge tube, discarding the magnetic beads.

[0173] 5.5 Add 0.2x volume of magnetic beads to the supernatant, mix well by pipetting up and down, and let it stand at room temperature for 8 minutes.

[0174] 5.6 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 5 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand. Discard the supernatant.

[0175] 5.7 Keep the PCR tube on the magnetic stand and add 220 μL of freshly prepared 80% ethanol to the tube without pipetting up and down the magnetic beads. Let it stand at room temperature for 40 seconds. Discard the supernatant.

[0176] 5.8 Repeat step 5.7.

[0177] 5.9 Keep the PCR tube on the magnetic stand and air-dry it at room temperature until the magnetic beads just start to crack (about 5 minutes).

[0178] 5.10 Remove the PCR tube from the magnetic stand and add no less than 20 μL of Nuclease-free Water or TE to elute the DNA. Mix well by pipetting up and down or vortexing, and let it stand at room temperature for 3 minutes.

[0179] 5.11 Place the PCR tube on the magnetic stand and let it stand at room temperature until the solution becomes clear (about 2 minutes) to allow the magnetic beads to fully adsorb onto the inner wall of the tube close to the magnetic stand.

[0180] 5.12 Transfer the eluate to a clean centrifuge tube, measure the concentration, and store it at -25 °C.

[0181] Comparative Example 1

[0182] The method for constructing the DNA library mainly includes the following steps: end repair and 3'-adenylation of cell-free DNA in pregnant women's peripheral blood; ligation of sequencing adapters and special tags; purification of DNA; different from the present invention, ordinary amplification is performed on the purified DNA; and ordinary purification is performed on the amplification products.

[0183] The fragment quality inspection chart is as Figure 1 shown, with a relatively wide fragment range and a relatively low total content.

[0184] All sequencing data in the present invention are aligned with the human genome reference sequence (version: NCBI Build37 / hg19) through the TMAP software. At the same time, we use the BamDuplicates command in the DA8600 sequencing platform to perform duplicate sequence removal analysis on the alignment results. After the above steps of analysis, the uniquely matched sequences with a mapping quality value (MAPQ) greater than 10 and a sequence length greater than 35 bp are extracted, and the locally weighted regression method is used to perform GC correction on the obtained uniquely matched sequences. Finally, the percentage of the Y chromosome after GC correction is calculated. The proportion of fetal cell-free DNA is approximately equal to the percentage of the Y chromosome. P < 0.05 indicates that the difference is statistically significant.

[0185] The test data of Comparative Example 1 were analyzed to obtain the results in Table 1, showing a relatively low content of fetal cell-free DNA (cffDNA).

[0186] Table 1: Content of cffDNA in pregnant women's peripheral blood

[0187]

[0188] Comparative Example 2

[0189] Based on Comparative Example 1, the fragment screening step in the method of the present invention is added. The main process includes: end repair and 3'-adenylation of cell-free DNA in pregnant women's peripheral blood; ligation of sequencing adapters and special tags; purification of DNA; normal amplification of the purified DNA; and fragment screening of the amplification products.

[0190] The fragment quality inspection chart is as Figure 2 . The results show that the DNA fragments after fragment screening are concentrated in the range of 200 - 300 bp, with a peak at about 260 bp.

[0191] After high-throughput sequencing, bioinformatics analysis obtained the content of cell-free fetal DNA (cffDNA) in Table 2, which is higher than that in Comparative Example 1.

[0192] Table 2: Content of cffDNA in pregnant women's peripheral blood

[0193]

[0194] Comparative Example 3

[0195] Bovine serum albumin and 1-butyl-3-methylimidazolium bromide ([BMIm]Br) are added during the PCR amplification process. The main process includes: end repair and 3'-adenylation of cell-free DNA in pregnant women's peripheral blood; ligation of sequencing adapters and special tags; purification of DNA; enhancer amplification of the purified DNA; and normal purification of the amplification products.

[0196] The fragment quality inspection chart is as Figure 3 , the fragment range is relatively wide, and the total content is higher than that in Comparative Example 1.

[0197] After high-throughput sequencing, bioinformatics analysis obtained the results in Table 3, and the content of cell-free fetal DNA (cffDNA) is still low.

[0198] Table 3: Content of cffDNA in pregnant women's peripheral blood

[0199]

[0200] Test Example 1

[0201] Based on Comparative Example 3, the fragment screening step is added. The DNA library construction method is optimized using two-step magnetic bead fragment sorting. The volume ratio in the first round (DNA Beads:DNA) is 4:5, and the volume ratio in the second round (DNA Beads:DNA) is 1:5. That is, all steps are the same as the construction method in Example 1 of the present invention. The main process includes: end repair and 3'-adenylation of cell-free DNA in pregnant women's peripheral blood; ligation of sequencing adapters and special tags; purification of DNA; enhancer amplification of the purified DNA; and fragment screening of the amplification products.

[0202] The fragment quality inspection diagram is as follows Figure 4 , the DNA fragment range is clear and the content has increased compared to Comparative Examples 2 and 3.

[0203] After high-throughput sequencing, the bioinformatics analysis obtained the results in Table 4, and the content of cell-free fetal DNA (cffDNA) was the highest.

[0204] Table 4: Content of cffDNA in pregnant women's peripheral blood

[0205]

[0206] Therefore, it can be seen that adding both an enhancer and a fragment screening step has the best effect on increasing the content of cell-free fetal DNA in the DNA library construction method for fetal chromosomal aneuploidy detection.

[0207] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0208] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention. All of these fall within the protection scope of the present invention.

Claims

1. A method for constructing a DNA library in the detection of fetal chromosomal aneuploidy, characterized in that, It includes the following steps: S1. Perform end repair and 3'-A addition on DNA; S2. Ligate sequencing adapters and tags; S3. Purify DNA; S4. Amplify the purified DNA; S5. Purify the amplified product by fragment screening.

2. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 1, wherein: In 1 - 5 ng of free DNA, use 2 μL of end repair enzyme and 10 μL of buffer, and perform blunt-end ligation and A-tailing on the free DNA in a PCR instrument.

3. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 2, characterized in that: The reaction program in the PCR instrument is: 4°C, 1 min; 32°C, 30 min; 65°C, 20 min; 4°C, Hold.

4. The DNA library construction method in the detection of fetal chromosomal aneuploidy according to claim 1, wherein: The method for ligating sequencing adapters and tags includes: Add 14 μL of T4 DNA ligase buffer, 3 μL of T4 DNA ligase, 5 μL of Adaptor, 5 μL of Index, and 3 μL of NF water to the reactant after the end of the S1 reaction, and the reaction program in the PCR instrument is 23°C, 20 min.

5. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 1, wherein: In S3, one of Ampure XP magnetic beads, KAPA Pure magnetic beads, VAHTS DNA Clean magnetic beads, ProNex magnetic beads, TIANSeq DNA fragment sorting magnetic beads, and MagicPure Size selection DNA magnetic beads is used for DNA purification.

6. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 5, characterized in that: The method for purifying DNA in S3 includes: 1) Equilibrate the magnetic beads in advance for 20 - 40 min; 2) Add 1x volume of magnetic beads to the ligation product, pipette and mix well, and let it stand at room temperature for 3 - 8 minutes; 3) Place the PCR tube on a magnetic rack and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the inner wall of the tube close to the magnetic rack, and discard the supernatant; 4) Keep the PCR tube on the magnetic rack, add 180 - 220 μL of freshly prepared 80% ethanol to the tube, let it stand at room temperature for 20 - 40 seconds, and discard the supernatant; 5) Repeat step 4); 6) Keep the PCR tube on the magnetic rack and air-dry it at room temperature until the magnetic beads crack; 7) Remove the PCR tube from the magnetic rack, add no less than 20 μL of NF Waer or TE to elute DNA, pipette and mix well, or vortex and mix well, and let it stand at room temperature for 1 - 3 minutes; 8) Place the PCR tube on the magnetic rack and let it stand at room temperature until the solution becomes clear, so that the magnetic beads are fully adsorbed on the inner wall of the tube close to the magnetic rack.

7. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy as described in claim 1, wherein: In S4, the PCR enhancers used for amplifying the purified DNA are bovine serum albumin and 1-butyl-3-methylimidazolium bromide.

8. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 7, wherein: The concentration of bovine serum albumin is 1 - 10 mg / mL, and the concentration of 1-butyl-3-methylimidazolium bromide is 1.5 mol / L.

9. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 7, characterized in that: The PCR amplification reaction program is: 98°C, 3 min; 98°C, 30 s; 60°C, 30 s; 72°C, 30 s; The number of cycles is set to 15; 72°C, 5 min; 4°C, Hold.

10. The method for constructing a DNA library in the detection of fetal chromosomal aneuploidy according to claim 1, wherein: The method for fragment screening in S5 includes: 1) Equilibrate the magnetic beads in advance for 20 - 40 min, and add 0.8x volume of magnetic beads to the amplified product; 2) Pipette and mix well, or vortex and mix well, and let it stand at room temperature for 3 - 8 minutes; 3) Place the PCR tube on a magnetic stand and let it stand at room temperature until the solution becomes clear, allowing the magnetic beads to fully adsorb onto the tube wall close to the magnetic stand. 4) Keep the PCR tube on the magnetic stand and transfer the supernatant to another clean centrifuge tube, discarding the magnetic beads. 5) Add magnetic beads with a volume of 0.2x to the supernatant, pipette and mix well, and let it stand at room temperature for 3 - 8 minutes. 6) Place the PCR tube on a magnetic stand and let it stand at room temperature until the solution becomes clear, allowing the magnetic beads to fully adsorb onto the tube wall close to the magnetic stand, and discard the supernatant. 7) Keep the PCR tube on the magnetic stand, add 180 - 220 μL of freshly prepared 80% ethanol to the tube, let it stand at room temperature for 20 - 40 seconds, and discard the supernatant. 8) Repeat step 7). 9) Keep the PCR tube on the magnetic stand and air-dry it at room temperature until the magnetic beads crack. 10) Remove the PCR tube from the magnetic stand, add no less than 20 μL of Nuclease-free Water or TE to elute the DNA, pipette and mix well, or vortex mix, and let it stand at room temperature for 1 - 3 minutes. 11) Place the PCR tube on a magnetic stand and let it stand at room temperature until the solution becomes clear, allowing the magnetic beads to fully adsorb onto the tube wall close to the magnetic stand. 12) Transfer the eluate to a clean centrifuge tube, measure the concentration, and store it at -15 to -25 °C.

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