Method for high-throughput screening of correct DNA molecules and application thereof

Abstract

The invention belongs to the technical field of high-throughput gene screening, and particularly relates to a method for high-throughput screening of correct DNA molecules and application of the method. The method comprises the following steps: S1, mixing to-be-screened synthetic products of target genes to obtain DNA pool which corresponds to the target genes and has the same DNA molecule number concentration; s2, the DNA pool is diluted; s3, adding the diluent into pores of a porous plate, and carrying out PCR amplification by taking DNA molecules in the diluent as a template; and S4, carrying out sequencing analysis on the PCR product, and screening to obtain correct DNA molecules. The method is low in cost and simple and convenient to operate, can realize high-throughput accurate screening of correct DNA molecules, is not limited by the size of the DNA molecules, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of high-throughput gene screening, and particularly relates to a method for high-throughput screening of correct DNA molecules and application thereof. BACKGROUND

[0002] Current mainstream Oligo synthesis technologies mainly include column synthesis and chip synthesis technologies, and they are essentially chemical synthesis. The side reactions accompanying chemical synthesis bring various mutations to the Oligo or Oligo pool. Therefore, the double-stranded DNA product synthesized by multiple Oligos through PCA (Polymerase Cycling Assembly) or other ways also contains various mutations. These double-stranded DNA products containing mutations need to be cut by restriction endonuclease, recovered by gel / cleanly recovered, connected by T4 ligase (or recombined), and then, after being circularized with linearized vectors, transformed, plated, 8-16 single colony PCRs, 2-4 bacterial culture, 2-4 plasmid extraction, 2-4 sequencing, and finally, the completely correct DNA molecules are obtained. As can be seen, a complete DNA screening process has multiple steps, which is time-consuming and costly. Therefore, a high-throughput gene screening technology is needed.

[0003] In the invention patent "A method for screening sequence-correct DNA molecules and application thereof", only one molecule (amplified from one molecule) can be screened in a single reaction system, and 10 molecules are screened for each gene. For 200 genes, 2000-hole reaction systems need to be prepared, and then, limited by the number of NGS adapter combinations (96 double-end unique indexes), 21 NGS sequencing reactions are needed to split the 2000-hole reaction systems (corresponding to 2000 molecules of 200 genes, 10 molecules per gene) for sequencing. This has a cost advantage over traditional molecular cloning, but it is still not simple and the cost is still too high; and only 10 molecules per gene are screened, which may have the following problems: a, the screened DNA contains mutations; b, due to uneven distribution, there are 2 molecules of a gene or no DNA molecules in a reaction system.

[0004] Current research results have reported high-throughput screening techniques for DNA (such as Schwartz, J. J., Lee, C. & Shendure, J. Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules. Nat. Methods 9, 913 (2012)), but the technique is limited by NGS read length, only short DNA (120 bp) can be screened, and a pair of specific tag amplification primers needs to be designed for each correct 120 bp molecule screened, greatly increasing the cost. SUMMARY

[0005] In view of the above technical problems, the present application provides a method for high-throughput screening of correct DNA molecules and its application. The method is low in cost, simple in operation, can realize high-throughput and accurate screening of correct DNA molecules, and is not limited by the size of DNA molecules.

[0006] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions: The first aspect of the present application provides a method for high-throughput screening of correct DNA molecules, which specifically comprises the following steps: S1, calculating the molecular concentration of DNA molecules of the synthesized product to be screened obtained by synthesizing target genes, mixing the synthesized product to be screened of each target gene according to the number of DNA molecules according to the molecular concentration, obtaining a DNA pool, and the molecular concentration of the DNA molecules obtained by synthesizing each target gene in the DNA pool is equal; S2, diluting the DNA pool to obtain a dilution solution, and the number of DNA molecule copies contained in 20 μL of the dilution solution is 0.9-1.1 times the number of target genes; S3, adding the dilution solution to the wells of a multi-well plate, and performing PCR amplification with the DNA molecules in the dilution solution as templates, and the number of DNA molecule copies in each well is 0.9-1.1 times the number of target genes; S4, recovering the PCR products of each well of S3, taking a part of the PCR products in each well for NGS sequencing, and analyzing the sequencing results to screen the PCR products with corresponding target gene sequence information and without mutations, and the template in the well corresponding to the PCR product is the correct DNA molecule.

[0007] In S1, the synthetic products are mixed according to the molecular number concentration of DNA to obtain a DNA pool in which the DNA molecules of any two target genes are the same. S1 has no limit on the number of target genes, and any number can be selected according to experimental needs, such as 100, 200, 500, 1000.

[0008] In S2, the ideal state after dilution is to contain 10 copy DNA molecules per microliter of solution, but in actual operation, it is not possible to dilute completely accurately. Taking 200 target genes (gene numbers 200-1, 200-2, 200-3,..., 200-199, 200-200) as an example, after continuous dilution, each 20 μL may contain 180 copy, 185 copy, 205 copy, 210 copy, 220 copy, but this does not affect the final screening effect. The dilution solution of the 200 target gene synthesis products is added to each well as a template in S3.

[0009] In S3, since the DNA molecules of the same target gene obtained by synthesis have the same amplification homologous arm at the synthesis site, these DNA molecules of different genes can be amplified and enlarged by the same pair of amplification primers.

[0010] In the sequencing result analysis of S4, the sequencing data of each well will appear the following four situations: (1) no sequence information of target genes; (2) sequence information of target genes, but containing mutations; (3) sequence information of target genes, not containing mutations; (4) sequence information of target genes, containing mutations and correct molecules. The third case (i.e., sequence information of target genes, not containing mutations) has and only has the correct DNA molecule of the corresponding gene in the well, which meets the requirements.

[0011] The present application ingeniously mixes DNA molecules of multiple target genes (such as 200 genes) into a DNA pool at equal concentration, and then dilutes the DNA pool to a very low concentration through multiple stages, and then distributes the DNA pool into multiple reaction wells (such as 96 wells), so that: (1) for a single gene, only one DNA molecule of the target gene is contained in each reaction well. Even if there are 0 or 2 DNA molecules, it does not affect the screening because it conforms to the Poisson distribution; (2) for a specified well, the well contains one DNA molecule from all the synthetic products of the target genes (or a small error, such as 0 or 2), if there are 200 target genes, then the well contains about 200 DNA molecules and each DNA molecule corresponds to a target gene; (3) if 96 wells are used for parallel reaction, then each target gene is screened for 96 molecules, which greatly simplifies the operation steps and reduces the cost. The correct retrieval rate of high-throughput screening by the method is high, and the correct DNA molecules missed in the screening can be supplemented by traditional molecular cloning, one-generation Sanger sequencing and other methods.

[0012] Preferably, the method for calculating the number concentration of molecules in S1 is: converting the mass concentration into the amount-of-substance concentration (such as ng / μL into pmol / μL) according to the molecular weight and mass concentration of each DNA, and then converting the amount-of-substance concentration into the number concentration of molecules (such as pmol / μL into copy / μL).

[0013] Preferably, the method for dilution in S2 is continuous dilution.

[0014] Preferably, the PCR amplification in S3 is performed using a high-sensitivity hot-start PCR mix.

[0015] Further preferably, the reaction system for the PCR amplification is: 2×PCR mix 25 μL, upstream primer 2.5 μL, downstream primer 2.5 μL, and the dilution solution obtained in S2 20 μL.

[0016] Preferably, the multi-well plate in S3 is a 96-well plate.

[0017] Optionally, in S4, different Index adapters can be added to the PCR products for data differentiation.

[0018] Preferably, the method further comprises obtaining a completely correct DNA product by PCR amplification using the correct DNA molecule obtained in S4 as a template. The prepared DNA product is a pure product, which can be used for further assembly of large plasmids or establishment of a pure plasmid library, and can also be connected to a single gene connection vector to reduce the sample number of colony PCR, shaking, plasmid extraction and sequencing.

[0019] In a second aspect, the present application provides application of the method for high-throughput screening of correct DNA molecules in synthesis of TCR-T library genes. The TCR-T library genes are a mixture of multiple long genes, and sequence accuracy is required.

[0020] In a third aspect, the present application provides application of the method for high-throughput screening of correct DNA molecules in high-throughput screening of basic fragments for synthesis of large plasmids. A large plasmid is composed of small DNA fragments, for example, a 200 kb large plasmid is composed of 200 1 kb small DNA fragments. By using the method provided by the present application, it is not necessary to link each basic fragment to a vector for screening, and only the completely correct DNA molecule needs to be screened for the next assembly.

[0021] The present application has the beneficial effect that the method for high-throughput screening of correct DNA molecules provided by the present application uses as few steps as possible and low cost to perform high-throughput screening of synthesis products of target genes. For screening of hundreds or even thousands of target genes, the method only needs to perform at most 96-well reactions and 1 NGS sequencing, and can screen out the completely correct DNA molecule from the synthesis products of the target genes, avoiding cumbersome screening steps such as restriction enzyme digestion, gel recovery / clean recovery, T4 ligase connection (or recombination), transformation, plating, single colony picking PCR, shaking, and plasmid extraction, and is simple to operate, low in cost, and not limited by DNA size (can be amplified), and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Schematic diagram of PCA products of each gene contained in each hole position in Example 1 of the present application Figure 2 Amplification effect in 48 holes in Example 1 Figure 3 NGS sequencing result in Example 1 Figure 4 Result of amplification and retrieval of 192 genes in Example 1 DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0024] Screening of correct DNA molecules in gene synthesis products often needs multiple operation steps, which is time-consuming and costly. Existing DNA screening technologies have the possibility of not screening correct DNA molecules, or have limitations on DNA length.

[0025] The embodiment of the present application provides a method for high-throughput screening of correct DNA molecules, which specifically comprises the following steps: S1, calculating the molecular concentration of DNA molecules of the synthesis product to be screened obtained by synthesizing target genes, mixing the synthesis products to be screened of each target gene according to the number of DNA molecules according to the molecular concentration, and obtaining a DNA pool, wherein the molecular concentration of the DNA molecules obtained by each target gene in the DNA pool is equal; S2, diluting the DNA pool to obtain a dilution solution, wherein the number of DNA molecule copies contained in 20 μL of the dilution solution is 0.9-1.1 times the number of target genes; S3, adding the dilution solution to the wells of a multi-well plate, and performing PCR amplification with the DNA molecules in the dilution solution as templates, wherein the number of DNA molecule copies in each well is 0.9-1.1 times the number of target genes; S4, recovering the PCR products of each well in S3, taking a part of the PCR products in each well for NGS sequencing, and analyzing the sequencing results to screen the PCR products with sequence information corresponding to the target genes and without mutations, wherein the template in the well corresponding to the PCR product is the correct DNA molecule.

[0026] The method is low in cost, simple in operation, can realize high-throughput and accurate screening of correct DNA molecules, and is not limited by the size of DNA molecules.

[0027] The scheme of the present application is described below through specific examples.

[0028] The reagents in the following examples are obtained from commercial sources or obtained according to the methods known in the art unless otherwise specified; the methods in the following examples are the methods known in the art unless otherwise specified.

[0029] Example 1 The embodiment of the present application provides a method for screening a specified sequence DNA from a mixed pool of 192 genes of about 800 bp. The 192 genes of about 800 bp are derived from sequences generated in actual production of gene synthesis, and are synthesized by a plurality of Oligo through PCA, numbered as 192-1, 192-2, …, 192-192.

[0030] Since the continuous dilution of the method is tens of thousands of times, the difference in gene length and quality has almost no effect on the number of molecules after dilution, which can be ignored. For easy calculation, the above 192 genes are uniformly calculated according to 800 bp, 500000.00 g / mol.

[0031] Step 1, concentration measurement The sample concentration was measured using Nanodrop.

[0032] Step 2, first dilution The PCA product of each gene was diluted to 5 ng / μL.

[0033] Step 3, equal mixing Take 50 ng (i.e. 10 μL) of the diluted PCA product of 192-1 gene to a new 1.5 ml centrifuge tube, take 50 ng of the diluted PCA product of 192-2 gene to the centrifuge tube,... take 50 ng of the diluted PCA product of 192-191 gene to the centrifuge tube, take 50 ng of the diluted PCA product of 192-192 gene to the centrifuge tube (as mentioned before, each gene is considered to be equal in length), and make a DNA pool, at this time the DNA concentration is 5 ng / μL, which is equivalent to about 10 fmol / μL, i.e. 6x10 9 copy / μL.

[0034] Step 4, second dilution According to the calculation, the DNA pool is diluted to 1.92x10 9 copy / μL: take 1000 μL of the DNA pool solution obtained in step 3, add to 2125 μL of 1 mM EDTA aqueous solution (pH 8.0), place at 4°C, and mix on a rotating mixer for 2 h; Step 5, third dilution According to the calculation, the DNA pool is diluted to 1920000 copy / μL: take 0.1 mL of the DNA pool solution prepared in step 4, add to 99.9 mL of 1 mM EDTA aqueous solution (pH 8.0). Place at 4°C, and mix on a rotating mixer for 2 h; Step 6, fourth dilution According to the calculation, the DNA pool is diluted to 1920 copy / μL: take 0.1 mL of the DNA pool solution prepared in step 5, add to 99.9 mL of 1 mM EDTA aqueous solution (pH 8.0). Place at 4°C, and mix on a rotating mixer for 2 h; Step 7, fifth dilution According to the calculation, the DNA pool was diluted to 19.2 copy / μL (192 copy / 10 μL): take 1 mL of the DNA pool solution prepared in step 6, add to 99 mL of 1 mM EDTA aqueous solution (pH 8.0). Place at 4℃, mix on a rotating mixer for 2 h; Step 8, 6th dilution According to the calculation, the DNA pool was diluted to 192 copy / 20 μL: take 50 mL of the DNA pool solution prepared in step 7, add to 50 mL of 1 mM EDTA aqueous solution (pH 8.0). Place at 4℃, stir on a magnetic stirrer for 16 h; Step 9, hole expansion Before the start of amplification, 0.1 mL of 1M MgCl2 solution was added to the DNA pool solution in step 8 and mixed to chelate EDTA, eliminating the inhibition of EDTA on the amplification system. On a 96-well plate, select 48 wells, add 2x PCR mix 25 μL, F primer 2.5 μL, R primer 2.5 μL, and add 20 μL of the 192 copy / 20 μL DNA dilution prepared in step 8 as template. Amplification program: 95℃, 5 min; 95℃, 30 s, 58℃, 30 s, 72℃, 1 min, 40 cycles; 72℃, 5 min.

[0035] The schematic diagram of the PCA products of each gene contained in each hole is shown in Figure 1 The 48-hole amplification effect is shown in Figure 2 Due to the different lengths of the 192 genes and the low template concentration, the bands are weak and multiple, which is a normal phenomenon.

[0036] Step 10, NGS sequencing The sequencing results were analyzed. Taking holes 1~hole 28 of 192-1~192-38 as an example, the analysis results of sequencing are shown in Figure 3 Green corresponds to a score of 100, indicating that there is and only the correct DNA molecule, white corresponds to a score <100, indicating that there is a deletion or mutation, the more the deletion and mutation, the lower the score; hole 2, 4, 8, 11, etc. corresponding to the 192-1 gene all contain and only contain the correct DNA molecule of the gene, any one of which can be selected as the correct DNA molecule of the gene amplified by the module.

[0037] Step 11, retrieval Design specific amplification primers to amplify the target DNA using the selected amplification tube as the template. The results of amplification retrieval of the 192 genes are shown in Figure 4 A total of 189 genes were retrieved, with a success rate of 98%: Step 12: re-verify the carrier After recovering the 189 genes amplified in step 11, each was ligated to pClone007 vector, and one transformant was picked from each for testing, and the accuracy was calculated, and the results are shown in Table 1.

[0038] Table 1: sequencing results

[0039] As can be seen from the results, the correct retrieval rate is 94.7%, the sequencing results of the amplified double bands have no obvious influence, and the screening is successful. For the missed DNA molecules, traditional molecular cloning methods, first-generation Sanger sequencing and other methods can be used for supplementary screening.

[0040] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of high throughput screening for correct DNA molecules, characterized in that, Specifically comprising the following steps: S1, calculating the molecular number concentration of DNA molecules of the synthesized target gene to obtain the synthesized product to be screened, mixing the synthesized product to be screened of each target gene according to the DNA molecular number concentration, obtaining a DNA pool, and the DNA pool has equal molecular number concentration of DNA molecules obtained by synthesizing each target gene; S2, diluting the DNA pool to obtain a dilution solution, and the DNA molecule copy number contained in 20 μL of the dilution solution is 0.9-1.1 times the number of target genes; S3, adding the dilution solution to the wells of a multi-well plate, and using the DNA molecules in the dilution solution as templates for PCR amplification, and the DNA molecule copy number in each well is 0.9-1.1 times the number of target genes; S4, recovering the PCR products of each well in S3, taking a part of the PCR products from each well for NGS sequencing, and analyzing the sequencing results to screen the PCR products with corresponding target gene sequence information and without mutations, and the template in the well corresponding to the PCR product is the correct DNA molecule.

2. The method of high-throughput screening for correct DNA molecules according to claim 1, characterized in that, The method for calculating the molecular number concentration in S1 is: converting the amount of substance concentration according to the molecular weight and mass concentration of each DNA, and then converting the amount of substance concentration to the molecular number concentration.

3. The method of high-throughput screening for correct DNA molecules according to claim 1, characterized in that, The method for dilution in S2 is continuous dilution.

4. The method of high-throughput screening for correct DNA molecules according to claim 1, characterized in that, The PCR amplification in S3 uses a high-sensitivity hot-start PCR mix.

5. The method of high-throughput screening for correct DNA molecules according to claim 4, characterized in that, The reaction system of the PCR amplification is: 2×PCR mix 25 μL, upstream primer 2.5 μL, downstream primer 2.5 μL, and 20 μL of the dilution solution obtained in S2.

6. The method of high-throughput screening for correct DNA molecules according to claim 1, wherein, The multi-well plate in S3 is a 96-well plate.

7. The method of high-throughput screening for correct DNA molecules according to any one of claims 1 to 6, characterized in that, The method further comprises using the correct DNA molecule obtained in S4 as a template to obtain a completely correct DNA product by PCR amplification.

8. The application of the method for high-throughput screening of correct DNA molecules in claim 1-7 in synthesizing TCR-T library genes.

9. The application of the method for high-throughput screening of correct DNA molecules in claim 1-7 in high-throughput screening of basic fragments for synthesizing large plasmids.

Images