A kit for whole genome amplification of bovine embryonic cells and application thereof

Abstract

This invention discloses a kit for amplifying the whole genome of bovine embryonic cells and its applications. The invention provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. This invention can utilize 4-8 bovine embryonic cells or genomic DNA down to the pg level to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs. Furthermore, the three-step operation process of this invention (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a kit for whole-genome amplification of bovine embryonic cells and its application. Background Technology

[0002] Embryo production and embryo transfer are rapid propagation techniques for dairy cattle, widely used in breeding and herd expansion. Producing high-quality embryos with high breeding value accelerates the propagation of superior genes, improving breeding efficiency and dairy herd productivity. Embryonic genome selection (EGS) technology provides a basis for early evaluation of embryos' breeding value and has gradually become a hot topic in bovine reproductive breeding research. EGS can easily help select embryos of ideal sex and superior genotypes for transfer; furthermore, it can eliminate embryos carrying recessive lethal genes or other chromosomal aberrations, reducing the frequency of lethal genes; and it can also accelerate the breeding process by increasing selection intensity and shortening generation intervals.

[0003] The key to bovine EGS is obtaining a sufficient amount of embryonic cell DNA without excessively damaging the embryo's subsequent developmental capacity. The amount of DNA obtained from the small number of embryonic cells obtained through embryo biopsy is insufficient for subsequent whole-genome sequencing, SNP genotyping, and other procedures. Therefore, whole-genome amplification technology is typically used to amplify the DNA from a small number of cells to obtain a sufficient amount of DNA.

[0004] MALBAC and MDA are two mainstream single-cell whole-genome amplification methods, each with its own technical characteristics and applicable scenarios. MALBAC technology aims to improve the uniformity of genome coverage by combining quasi-linear amplification with subsequent PCR amplification, making it more advantageous in copy number variation analysis. However, the polymerase used in its PCR amplification stage has relatively low fidelity, which may lead to a higher false positive rate for single nucleotide variants.

[0005] MDA technology relies on the high-fidelity phi29 DNA polymerase for isothermal amplification, which has a low error rate and performs better in applications requiring high fidelity, such as single nucleotide polymorphism analysis.

[0006] Although there are several commercial single-cell WGA kits based on the above technologies, they generally have some common problems and challenges. (1) Species-specific bias: The random primers used are designed based on human genome sequences, which may lead to significant bias when amplifying dairy cow and beef cow embryonic cells; (2) Amplification bias: Neither exponential amplification of MDA nor quasi-linear amplification of MALBAC combined with PCR can completely eliminate sequence-dependent amplification efficiency differences. Some genomic regions (such as high GC or highly repetitive regions) may be under-amplified or missing, affecting the accuracy and comprehensiveness of subsequent analysis; (3) Errors introduced during amplification: All WGA technologies inevitably introduce errors (such as point mutations, small fragment insertions / deletions) when performing large-scale DNA replication. Although the high-fidelity Phi29 polymerase can significantly reduce the error rate, the problem still exists. These errors may interfere with the identification of real genetic variations, especially when performing high-level SNP analysis; (4) Cost: The price is generally high, which limits its use in agricultural breeding research. Summary of the Invention

[0007] The purpose of this invention is to provide a kit for whole-genome amplification of bovine embryonic cells and its application.

[0008] Firstly, this invention claims protection for a primer set.

[0009] The primer set claimed in this invention is primer set 1 or primer set 2;

[0010] The primer set 1 consists of 20 primers as shown in A1)-A20) below; Primer set 2 consists of 10 primers as shown in A1)-A10) below; A1) Primer 1; Primer 1 has the nucleotide sequence AGTGAC; A2) Primer 2; Primer 2 has a nucleotide sequence of AAGCTT; A3) Primer 3; Primer 3 has a nucleotide sequence of CATGGC; A4) Primer 4; Primer 4 has the nucleotide sequence CTGACC; A5) Primer 5; Primer 5 has a nucleotide sequence of CATAGA; A6) Primer 6; Primer 6 has a nucleotide sequence of GAATCC; A7) Primer 7; the nucleotide sequence of primer 7 is ATCTTG; A8) Primer 8; Primer 8 has a nucleotide sequence of CATTGA; A9) Primer 9; Primer 9 has the nucleotide sequence ATCTGG; Primer 10 (A10); Primer 10 has a nucleotide sequence of GTCAAA; A11) Primer 11; the nucleotide sequence of primer 11 is CTAAGA; Primer 12 (A12); the nucleotide sequence of primer 12 is GGCTGA; A13) Primer 13; the nucleotide sequence of primer 13 is TGCCCA; Primer 14 (A14); Primer 14 has a nucleotide sequence of GCTCCA; Primer 15 (A15); Primer 15 has a nucleotide sequence of AGATGC; Primer 16 (A16); the nucleotide sequence of primer 16 is ATAGCA; Primer 17 (A17); Primer 17 has the nucleotide sequence ACTGGG; Primer 18 (A18); the nucleotide sequence of primer 18 is AGCCTC; Primer 19 (A19); Primer 19 has the nucleotide sequence GGGTCA; Primer 20 (A20); the nucleotide sequence of primer 20 is AGATAC.

[0011] In some embodiments of the present invention, the primer set is used for bovine whole genome amplification. Specifically, bovine genomic DNA is used as a template, specifically 20 ng of bovine genomic DNA.

[0012] In some embodiments of the present invention, the primer set is used for whole-genome amplification of bovine embryonic cells. Specifically, bovine embryonic cell lysis products are used as templates, specifically 6-10 bovine embryonic cell lysis products.

[0013] The primers in the primer set can be packaged individually or mixed in an equimolar ratio (e.g., each primer is diluted to the same concentration with sterile deionized water and mixed in equal volumes to prepare a primer mix for use).

[0014] Secondly, the present invention claims protection for reagents or kits containing the primer set described in the first aspect above.

[0015] In some embodiments of the present invention, the reagents or kits are used for bovine whole-genome amplification. Specifically, bovine genomic DNA is used as a template, specifically 20 ng of bovine genomic DNA.

[0016] In some embodiments of the present invention, the reagents or kits are used for whole-genome amplification of bovine embryonic cells. Specifically, bovine embryonic cell lysis products are used as templates, specifically lysis products from 6-10 bovine embryonic cells.

[0017] When the kit is used for whole-genome amplification of bovine embryonic cells, the kit also contains Phi29 DNA polymerase. Furthermore, the kit also contains a lysis buffer and / or a neutralizing solution. The lysis buffer is used to lyse bovine embryonic cells to release intracellular nucleic acids, and the neutralizing solution is used to terminate the lysis reaction of the lysis buffer to maintain the stability of subsequent nucleic acid processing steps.

[0018] In some embodiments of the present invention, the solvent of the lysis buffer is nuclease-free water, and the solutes and concentrations are 0.1M KOH and 2.5mM EDTA.

[0019] In some embodiments of the present invention, the neutralizing solution is prepared by mixing equal volumes of 400 mM Tris-HCl solution (pH 4.0) and 15 mM MgCl2 solution.

[0020] Thirdly, the present invention claims protection for the use of the primer set described in the first aspect above or the reagent or kit described in the second aspect above in any of the following: B1) Amplification of the whole genome of bovine embryonic cells; B2) Amplify the bovine genome.

[0021] Further, in step B2), the amplification of the bovine whole genome can be performed using bovine genomic DNA (especially trace amounts of DNA, such as 20 ng of bovine genomic DNA) as a template, or using lysate of bovine embryonic cells (such as lysate of 6-10 bovine embryonic cells) as a template.

[0022] Fourthly, this invention claims protection for a method for amplifying the whole genome of bovine embryonic cells, comprising the following steps: C1) Take 6-10 (e.g., 6-8, or even 4-8) bovine embryonic cells, first add the lysis buffer described in the second part above for lysis, and then add the neutralization buffer described in the second part above to terminate the lysis; C2) Using the system treated in step C1) as a template, PCR amplification was performed using Phi29 DNA polymerase and the primer set described in the first aspect above to obtain the amplification product, thus realizing the amplification of the whole genome of bovine embryonic cells.

[0023] Further, in step C1), 3 μL of the lysis buffer is added to 4 μL of preservation solution containing 6-10 bovine embryonic cells.

[0024] Further, in step C1), the pyrolysis conditions can be pyrolysis at 23-27°C (e.g., 25°C) for 5 minutes.

[0025] Furthermore, in step C1), the volume of the neutralizing solution added is the same as the volume of the pyrolysis solution added.

[0026] Further, in step C2), the final concentration of the primer set in the amplification system can be 50 μM (the total concentration after equimolar mixing of each primer is 50 μM).

[0027] Furthermore, in step C2), the reaction system for the PCR amplification is as follows: 2 μL of Phi29 DNA polymerase; 4 μL of 10×Phi29 reaction buffer; 10 μL of template; 20 μL of the primer set (working concentration 50 μM); 3.2 μL of dNTPs (working concentration 2 mM); and 0.8 μL of nuclease-free water.

[0028] Further, in step C2), the reaction program for the PCR amplification is as follows: 30℃ for 3 hours; 65℃ for 10 minutes; and incubation at 4℃. PCR instrument auxiliary settings: The PCR instrument heating cap temperature is set to 70℃.

[0029] Fifthly, the present invention claims protection for the application of the method described in the fourth aspect above in any of the following: D1) Next-generation sequencing; D2) Targeted capture sequencing.

[0030] In the foregoing related aspects, the cattle may be Holstein cattle. In some embodiments of the present invention, the bovine embryonic cells are embryonic cells of Holstein bulls.

[0031] Experiments demonstrate that this invention, using bovine reference genome sequences to design random primers and based on MDA whole-genome amplification technology with optimized reaction systems, can amplify high-coverage, low-mismatch-rate whole-genome DNA amplification products from 4-8 bovine embryonic cells or even pg-level genomic DNA. This amplification is directly compatible with downstream applications such as next-generation sequencing and targeted capture sequencing, while simultaneously reducing detection costs. Furthermore, the invention's three-step process (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours. Attached Figure Description

[0032] Figure 1 Agarose gel electrophoresis image for whole genome amplification. Lane AC: 50 μM primer final concentration; Lane DF: 20 μM primer final concentration; Lane GI: 2 μM primer final concentration; Lane M: 10k Marker. Detailed Implementation

[0033] The core technical steps and key design principles of bovine embryonic cell whole genome amplification in this invention are as follows: (1) Based on the bovine reference genome sequence (UMD_3.1.1 addv2 version), 6-base random primers were designed to ensure the randomness and uniformity of the primer binding sites in the genome, so as to achieve full-length unbiased amplification. The primer length needs to achieve a balance between random binding ability and specificity, and avoid the occurrence of multiple consecutive identical bases (such as GGGGGG) or simple repetitive sequences, so as to reduce non-specific binding and amplification deviation. Considering that the theoretical amplification length of Phi29 DNA polymerase can reach 70 kb, the average distance between primers should be much smaller than this value to ensure the effective amplification of long fragments.

[0034] (2) High-fidelity Phi29 DNA polymerase was used to achieve uniform amplification of the whole genome, with an average amplification product length >10 Kb.

[0035] (3) Cell lysis: Cell lysis is the basis for subsequent experiments. The goal is to maintain the integrity and amplification of genomic DNA to the maximum extent while completely destroying the cell structure.

[0036] The three-step procedure (cell lysis - lysis termination - isothermal amplification) can complete efficient amplification within 3.5 hours.

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Example 1: Design of random primers and verification of amplification effect This embodiment will involve random primers and use bovine genomic DNA as a template to verify the whole-genome amplification effect.

[0040] I. Design of Random Primers Based on the bovine Bos_taurus_UMD_3.1.1 addv2 genome (https: / / ngdc.cncb.ac.cn / gwh / ncbi_assembly / 34441 / show), 6 bp random primers were designed to ensure random binding of primers in the genome, which helps to achieve broad coverage of the whole genome and is particularly suitable for whole genome amplification.

[0041] First, the frequency of 6 bp sequences in the genome was statistically analyzed using the bioinformatics tool Jellyfish, forming a primer library. This library contained information on all 6 bp sequences in both the positive and negative strands of the genome, along with their frequency of occurrence. Considering primer specificity and coverage, primers with frequencies falling within the middle range were selected. Primers with perfectly inverse complementary sequences were removed, resulting in 477 6 bp sequences. The positions of these 477 6 bp sequences in a reference genome were then analyzed, divided into intervals, and the number of primers in each interval was counted. Primers with the highest frequency of occurrence in the genome were selected, and the library was then rigorously filtered and selected based on predefined parameters.

[0042] The filtering parameters are as follows: Each primer contains four bases to ensure uniform Tm values ​​and avoid non-specific binding due to high GC or weak binding due to low GC. Random base distribution is essential to prevent polypurine or polypyrimidine sequences. Self-complementarity should be minimized during design to avoid hairpin structures. Primer complementarity should also be reduced to prevent primer dimer formation. Considering the theoretical amplification length of Phi29 DNA polymerase can reach 70 kb, the spacing between primers must be much smaller than this. Whole-genome simulation using bioinformatics software ensures effective amplification of all genomic regions without significant bias.

[0043] The initial primer design interval was 19 kb, and 10 primer sequences were selected. The sequence information of the 10 primers and the genome coverage of the amplification products are shown in Tables 1 and 2.

[0044] Table 1. Information on 10 random primer sequences

[0045] Table 2. Genomic coverage information of the amplification products of 10 primers

[0046] II. Verification of Random Primer Amplification Effect 1. Test Objective (1) Determine the optimal primer concentration: Find the optimal primer concentration suitable for the current experimental system within the normal range of 20-75 μM.

[0047] (2) Assess the feasibility of low concentration (2 μM): Verify whether low primer input will lead to amplification failure, providing a basis for low-cost or special experiments.

[0048] Note: 20-75 μM is the working concentration range of common commercially available kits. The primer concentrations in the following tests are the sum of the concentrations of each primer in the primer combination.

[0049] 2. Whole genome amplification The amplification system is shown in Table 3.

[0050] Table 3. Whole Genome Amplification Reaction System

[0051] Note: "phi29 DNA polymerase" and "10×phi29 reaction buffer" are from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 14404ES80. "Random primers" are obtained by mixing equimolar amounts of the 10 primers in Table 1.

[0052] Whole genome amplification reaction program: 30℃, 3h, 65℃, 10min, 4℃ incubation. PCR instrument heating cap temperature set to 70℃.

[0053] The reaction conditions were uniform: a fixed amount of template was used (20 ng bovine genomic DNA).

[0054] The results showed that, under this experimental system, a primer concentration of 50 μM yielded the highest amplification yield, averaging 3.6 μg. While amplification was possible at a concentration of 20 μM, the yield was only about half that of the higher concentration, approximately 1.6 μg. A concentration of 2 μM was too low for practical whole-genome amplification experiments. Therefore, the final primer concentration in subsequent amplification tests was determined to be 50 μM. (See Table 4 and...) Figure 1 As shown.

[0055] Table 4. Concentration of whole-genome amplification products with different primer input amounts

[0056] 3. Whole-genome amplification product capture and sequencing All whole-genome amplification products obtained from the above steps were uniformly captured using a PHR0163_Bt40K_V1.0 Panel (44383 loci, Borui Biotechnology Co., Ltd.). The capture process was performed according to the instructions of the company's GenoBaits® DNA Hybridization and Wash Kit for Illumina (catalog number GB000600). The sequencing data volume per sample after capture was 1.5 G to ensure the reliability of the evaluation results.

[0057] The experiment also included a control group with normal DNA library construction. This control group used a common DNA library construction method in next-generation sequencing, involving restriction enzyme digestion and end repair of the DNA template, the addition of sequencing adapters, and finally the addition of sequencing barcodes to complete the final library construction. Specific instructions for library construction can be found in the GenoBaits® DNA Library Prep Kit for Illumina (catalog number GB000104) from Bored Biotechnology Co., Ltd. The normal library construction products obtained in this control group were also captured using the PHR0163_Bt40K_V1.0 Panel (44383 sites), with a single sample sequencing data volume of 1.5 G.

[0058] The results are shown in Table 5. The data performance of the 50 μM primer with better amplification effect at the final concentration is significantly different from that of normal DNA library construction, and has not yet reached the ideal data level. The primers need to be optimized again.

[0059] Table 5. Probe capture data of whole genome amplification products

[0060] Note: Normal DNA library construction refers to the data generated by the library construction and hybridization capture process using the Boredi DNA library construction kit with an input of 200 ng of bovine genomic DNA (the same applies below).

[0061] 4. Further screening and validation of random primers To improve the randomness and uniformity of random primer binding and lay a better foundation for subsequent whole-genome amplification, the primers were optimized. First, the amplification length in the primer design was shortened from 19 kb to 10 kb to increase primer density and cover weak regions with amplification. Second, the number of random primer sequences was increased from 10 to 20.

[0062] The optimized primer sequence information and the genome coverage information of the amplification products are shown in Tables 6 and 7.

[0063] Table 6. Information on the 20 optimized random primer sequences

[0064] Table 7. Genomic coverage information of 20 primer amplification products

[0065] Using steps 2 and 3 above (final primer concentration 50 μM, DNA input 20 ng), bovine whole genome amplification and amplification product capture and sequencing were performed using optimized random primers (20 primers in Table 6).

[0066] The results are shown in Table 8. Sequencing data indicate that under the existing amplification system, after primer optimization, the Uniformity_rate increased from 84.9% to 98.84%, and the SNP detection rate increased from 92.4% to 99.86%, which is close to the level of normal DNA library construction, reflecting to some extent the effectiveness of random primers and amplification system.

[0067] Table 8. Probe capture data of whole genome amplification products after primer optimization

[0068] Example 2: Whole genome amplification of bovine embryonic cells In this embodiment, bovine embryonic cells are used to further verify the random primers screened in Example 1, and the lysis buffer and neutralization buffer are optimized.

[0069] The embryonic cell samples used in this embodiment are all Holstein bull embryonic cells from Shijiazhuang Tianquan Fine Breed Dairy Cattle Co., Ltd. (Bull embryo refers to a calf born after the transplantation of this embryonic cell that is male, and the parents are Holstein cattle).

[0070] I. Preliminary testing of the 20 random primers of this invention using commercially available lysis buffer. Test samples: 4 embryonic cell samples, each with a volume of 4 μL, containing 6-10 bovine embryonic cells, which were stored in calcium and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500).

[0071] 1. Lysis of bovine embryonic cells Lysis buffer: Yikang Gene ChromLong TM The component in the Single-Cell Whole Genome Amplification Kit, catalog number KT100805624, is used as follows: Take 2.7 μL of the lysis buffer from the kit, add 0.3 μL of DTT, and mix to obtain the lysis buffer.

[0072] Neutralization solution: as shown in Table 9.

[0073] Table 9. Neutralization solution formulation of the present invention (20 μL)

[0074] Note: The solvents for Tris-HCl (400 mM, pH 4.0) and MgCl2 (15 mM) are both nuclease-free water.

[0075] The specific pyrolysis steps are as follows: (1) Add 3 μl of lysis mixture to each PCR tube (cell volume 4 μl), gently tap the tube wall to mix, and then centrifuge for 3-5 seconds. Note: Do not touch the liquid surface when adding lysis mixture, and do not shake vigorously to prevent cell sample loss.

[0076] (2) Place the PCR tube in the PCR instrument, select "On, 70℃" for "heat cap", and lyse at 65℃ for 10 min. (3) After the reaction is complete, add 3 μl of neutralization solution, mix well and place on ice for later use.

[0077] 2. Whole genome amplification and amplification product capture and sequencing Using the bovine embryonic cell lysis products obtained in step 1 as a template, whole-genome amplification was performed according to step 2 of Example 1 (final primer concentration was 50 μM), and the amplified products were captured and sequenced according to step 2 of Example 1. The normal DNA library preparation group, serving as a control, was operated in the same manner as in Example 1.

[0078] The results are shown in Table 10. The amplification uniformity, on-target performance, and SNP detection rate of the random primers in this invention reached a good level, indicating that the method is feasible. However, there is still a certain gap compared with the uniformity of normal DNA library construction and the SNP detection rate. This indicates that the current WGA workflow based on cell lysis still needs optimization in terms of amplification uniformity and the integrity of genetic variation detection.

[0079] Table 10. Probe capture data of bovine embryonic cell whole genome amplification products

[0080] II. Optimization of pyrolysis buffer and neutralization solution Three candidate protocols were set up, with three replicates for each protocol. The results were averaged. Bovine embryonic cells were used as test samples. Each test sample had a volume of 4 μL and contained 6-8 bovine embryonic cells. The bovine embryonic cells were stored in calcium and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500).

[0081] Option 1: Lysis buffer: sterile deionized water.

[0082] No neutralizing solution.

[0083] Option 2: Lysis buffer: Yikang Gene ChromLong TM The component in the Single-Cell Whole Genome Amplification Kit, catalog number KT100805624, is used as follows: Take 2.7 μL of the lysis buffer from the kit, add 0.3 μL of DTT, and mix to obtain the lysis buffer.

[0084] Neutralization solution: as shown in Table 9.

[0085] Option 3: The formulations of the lysis buffer are shown in Table 11, and the formulations of the neutralization buffer are shown in Table 9.

[0086] Table 11. Formulation of the lysis buffer of the present invention (1 mL)

[0087] Note: The solvents for KOH (1M) and EDTA (0.5M) are both nuclease-free water.

[0088] 1. Lysis of bovine embryonic cells Option 1: Add 6 μl of sterile deionized water to 4 μl of cell sample, gently tap to mix, centrifuge, incubate at 105°C or 98°C for 10 min in a PCR instrument to complete lysis, and place on ice for later use.

[0089] Option 2: Add 3 μL of the corresponding lysis buffer to each 4 μL test sample of bovine embryonic cells, mix well, place the PCR tube in the PCR instrument, select "On, 70℃" for "Heat Cap", and lyse at 65℃ for 10 min. After the reaction is complete, add 3 μL of neutralization solution, mix well, and place on ice for later use.

[0090] Option 3: Add 3 μL of the corresponding lysis buffer to each 4 μL test sample of bovine embryonic cells and lyse at 25°C for 5 min. Then add 3 μL of the corresponding neutralization buffer to the lysed cell mixture, mix well, and set aside.

[0091] 2. Whole genome amplification and amplification product capture and sequencing Using the bovine embryonic cell lysate obtained in step 1 as a template, whole-genome amplification was performed according to step 2 of Example 1 (final primer concentration was 50 μM), and the amplification products were captured and sequenced according to step 3 of Example 1. The normal DNA library preparation group, serving as a control, was operated in the same manner as in Example 1.

[0092] The results are shown in Table 12. The test results clearly show that the self-developed lysis scheme (Scheme 3) of this invention, combined with the existing amplification system (Table 3, final primer concentration of 50 μM), performs best. Its key indicators, Uniformity_rate and SNP detection rate, are 96.43% and 96.9%, respectively, which are close to the level of normal DNA library construction. This proves that the scheme has good compatibility with the existing amplification system.

[0093] Table 12. Probe capture data of whole genome amplification products using different lysis buffer schemes

[0094] III. Scale-up Sample Size Testing of Lysis Buffer To verify the stability of the self-developed lysis and neutralization scheme (Scheme 3) of this invention, 10 bovine embryonic cell samples were tested. Each sample had a volume of 4 μL and contained 6-8 bovine embryonic cells. The bovine embryonic cells were stored in calcium and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500). The specific operation was the same as Scheme 3 in step two.

[0095] The results are shown in Table 13. In amplification tests of 10 bovine embryonic cells, the self-developed lysis scheme of this invention achieved an average uniformity rate of 96.25%, with a maximum of 97.37%; the SNP detection rate was close to 97%, demonstrating stable and good performance. This indicates that the lysis scheme 3 determined in step two above exhibits stable lysis effects and is well-suited to existing amplification systems.

[0096] Table 13. Probe capture data of whole genome amplification products from the self-developed lysis scheme (Scheme 3) of this invention.

[0097] Through the testing in this embodiment, the kit for whole-genome amplification of bovine embryonic cells of the present invention was obtained, comprising: 20 primers in Table 6; Phi29 DNA polymerase; lysis buffer shown in Table 11 and neutralization buffer shown in Table 9.

[0098] Example 3: Comparison test of the whole genome amplification kit of the present invention with kits from other manufacturers. To compare the effectiveness of the self-developed reagent kit of this invention with other reagent kits, this embodiment tests the reagent kit of this invention with reagent kits from four well-known manufacturers on the market.

[0099] In this test, three embryonic cell samples from each manufacturer were tested. Each sample had a volume of 4 μL and contained 6-10 bovine embryonic cells. The bovine embryonic cells were stored in calcium- and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500). The operation method of the kit of this invention is described in Example 2 (corresponding to Scheme 3). For specific operation of commercially available kits, please refer to the corresponding instruction manual.

[0100] The results are shown in Table 14. The test data demonstrate that the kit of this invention exhibits excellent performance in core performance indicators. The average uniformity rate of its amplification products reached 96.67%, and the SNP detection rate was 96.94%, both superior to comparable commercially available kits. Furthermore, the cost of the kit of this invention is only 1 / 6 to 1 / 12 of that of similar commercially available products, demonstrating excellent cost-effectiveness while achieving high performance. Regarding stability, all replicate samples were successfully amplified, while some comparative commercially available kits experienced amplification failures, further proving the higher reliability of the kit of this invention.

[0101] Table 14. Probe capture data of whole genome amplification products from different manufacturers

[0102] Note: Manufacturer A's kit is a product of Nanjing Novizan, catalog number SC301-01. Manufacturer K's kit is a product of Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number CW2843S. Manufacturer C's kit is a product of Ibotek, catalog number RK21006. Manufacturer T's kit is a product of Thermo Scientific™, catalog number A39390.

[0103] Example 4: Comparative test of different samples from the same embryo using the kit of the present invention. To evaluate the amplification accuracy and reproducibility of the kit of this invention, this embodiment used three independent cell samples from the same embryo for parallel testing. Each sample volume was 4 μL, containing 6-8 bovine embryonic cells, which were stored in calcium- and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500). All samples underwent whole-genome amplification using the kit of this invention, captured using the PHR0163_Bt40K_V1.0 Panel, and the sequencing data volume per sample was controlled at 1.5 G. The specific operation was the same as in Example 2 (corresponding to Scheme 3). The stability of this kit was verified by comparing the consistency of key indicators among the three samples.

[0104] The test results are shown in Table 15. These results fully demonstrate that the kit of this invention possesses excellent reproducibility and high accuracy. Three parallel samples (sample 1, sample 2, and sample 3) from the same embryo showed highly consistent key indicators with minimal fluctuations. The test results of the three samples were highly close to the data from normal DNA library construction, proving that the kit of this invention can accurately and comprehensively restore genomic information at the microcellular level. Furthermore, the genotype consistency rate of the three samples reached 98.36%, further demonstrating the accuracy of amplification.

[0105] Table 15. Probe capture data of whole genome amplification products from different samples of the same embryo

[0106] In addition, the kit of this invention was used to amplify a small amount of bovine genomic DNA (2 ng genomic DNA) for whole-genome amplification. The DNA was captured using the PHR0163_Bt40K_V1.0 Panel, and the single-sample sequencing data volume was controlled at 1.5 G. The specific operation was the same as in Example 1 (corresponding to section II, verification of random primer amplification effect, with the DNA input amount changed from 20 ng to 2 ng, and the primer working concentration at 50 μM). The results are shown in Table 16.

[0107] Table 16. Data on probe capture of micro-volume bovine genomic DNA amplification products using the kit of the present invention.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A primer set, characterized in that: The primer set is primer set 1 or primer set 2; The primer set 1 consists of 20 primers as shown in A1)-A20) below; Primer set 2 consists of 10 primers as shown in A1)-A10) below; A1) Primer 1; Primer 1 has the nucleotide sequence AGTGAC; A2) Primer 2; Primer 2 has a nucleotide sequence of AAGCTT; A3) Primer 3; Primer 3 has a nucleotide sequence of CATGGC; A4) Primer 4; Primer 4 has a nucleotide sequence of CTGACC; A5) Primer 5; Primer 5 has a nucleotide sequence of CATAGA; A6) Primer 6; Primer 6 has a nucleotide sequence of GAATCC; A7) Primer 7; the nucleotide sequence of primer 7 is ATCTTG; A8) Primer 8; Primer 8 has a nucleotide sequence of CATTGA; A9) Primer 9; Primer 9 has the nucleotide sequence ATCTGG; Primer 10 (A10); Primer 10 has a nucleotide sequence of GTCAAA; A11) Primer 11; the nucleotide sequence of primer 11 is CTAAGA; Primer 12 (A12); the nucleotide sequence of primer 12 is GGCTGA; A13) Primer 13; the nucleotide sequence of primer 13 is TGCCCA; Primer 14 (A14); Primer 14 has a nucleotide sequence of GCTCCA; Primer 15 (A15); Primer 15 has a nucleotide sequence of AGATGC; Primer 16 (A16); the nucleotide sequence of primer 16 is ATAGCA; Primer 17 (A17); Primer 17 has the nucleotide sequence ACTGGG; Primer 18 (A18); the nucleotide sequence of primer 18 is AGCCTC; Primer 19 (A19); Primer 19 has the nucleotide sequence GGGTCA; Primer 20 (A20); the nucleotide sequence of primer 20 is AGATAC.

2. A reagent or kit containing the primer set of claim 1.

3. The reagent kit according to claim 2, characterized in that: The kit also contains Phi29 DNA polymerase.

4. The reagent kit according to claim 3, characterized in that: The kit also contains lysis buffer and / or neutralization buffer.

5. The reagent kit according to claim 4, characterized in that: The solvent of the lysis buffer is nuclease-free water, and the solutes and their concentrations are 0.1M KOH and 2.5mM EDTA; and / or The neutralization solution is prepared by mixing equal volumes of 400 mM Tris-HCl solution (pH 4.0) and 15 mM MgCl2 solution.

6. The use of the primer set of claim 1 or the reagent or kit of any of claims 2-5 in any of the following: B1) Amplification of the whole genome of bovine embryonic cells; B2) Amplify the bovine genome.

7. A method for amplifying the whole genome of bovine embryonic cells, comprising the following steps: C1) Take 6-10 bovine embryonic cells, first add the lysis buffer described in claim 4 or 5 to lyse them, and then add the neutralization buffer described in claim 4 or 5 to terminate the lysis. C2) Using the system treated in step C1) as a template, PCR amplification is performed using Phi29 DNA polymerase and the primer set described in claim 1 to obtain the amplification product, thereby realizing the amplification of the whole genome of bovine embryonic cells.

8. The method according to claim 7, characterized in that: In step C1), 3 μL of the lysis buffer is added to 4 μL of preservation solution containing 6-10 bovine embryonic cells; and / or, the lysis is performed at 23-27°C for 5 min; and / or, the volume of the neutralizing solution added is the same as the volume of the lysis buffer added. Furthermore, the preservation solution is a calcium and magnesium ion-free DPBS solution.

9. The method according to claim 7 or 8, characterized in that: In step C2), the final concentration of the primer set in the amplification system is 50 μM.

10. The application of the method according to any one of claims 7-9 in any of the following: D1) Next-generation sequencing; D2) Targeted capture sequencing.

Images