Primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof

Abstract

The present application relates to a primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof, and the primer combination is composed of 20 or 30 primers. The present application designs random primers based on sheep reference genome sequence, optimizes the reaction system based on MDA whole genome amplification technology, so that the whole genome DNA amplification product with high coverage and low mismatch rate can be generated by using 10 sheep embryo cells or as low as pg level genomic DNA, which can be directly adapted to downstream applications such as second-generation sequencing and targeted capture sequencing, and the detection cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding technology, specifically to primer combinations for amplifying the whole genome DNA of sheep embryonic cells and their applications. Background Technology

[0002] Genome selection (GS) utilizes whole-genome marker mapping to predict the early genetic value of individuals without waiting for phenotypic data, significantly advancing animal breeding processes, particularly in improving low heritability and limited traits. However, traditional GS suffers from a time lag between birth and genotype detection, limiting further reduction in generation intervals and becoming a bottleneck in its application. Embryonic genomic selection (EGS), a key strategy to overcome this bottleneck, maximizes selection intensity and reduces pregnancy and feeding costs for low-genetic-value embryos by performing biopsies, genotype detection, and calculating the estimated genetic breeding value (GEBV) of pre-implantation embryos. It has already been successfully applied in bovine breeding; for example, combining bovine blastocyst biopsy with low-starting-volume DNA sequencing and genotype filling techniques enables accurate prediction of GEBV and higher pregnancy rates. Chinese invention patent CN122128299A discloses a kit and application for amplifying the whole genome of bovine embryonic cells. It provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. It can amplify the whole genome DNA amplification product with high coverage and low mismatch rate using 4-8 bovine embryonic cells or down to pg level genomic DNA. It can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs.

[0003] In contrast, sheep EGS research is still in its early stages, facing three major technical obstacles: first, the efficiency of in vitro embryo production (IVEP) is low, making it difficult to meet the needs of large-scale biopsies; second, the window period for embryo biopsy and cryopreservation is unclear, and the impact of biopsy procedures on embryo viability has not been quantified; and third, there is a lack of standardized whole-genome amplification (WGA) platforms for sheep embryo biopsy samples. The key to sheep 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 segmented through embryo biopsy is insufficient for subsequent whole-genome sequencing, SNP genotyping, and other related work.

[0004] If a small number of sheep embryonic cells can be used to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate, and then conduct downstream DNA library construction and microarray detection, it will provide efficient technical support for accurate genetic evaluation of sheep pre-implanted embryos, shorten the breeding generation interval, and accelerate genetic progress. Summary of the Invention

[0005] To address the above problems, this invention proposes primer combinations for amplifying the whole genome DNA of sheep embryonic cells and their applications.

[0006] The primer combination for amplifying the whole genome DNA of sheep embryonic cells proposed in this invention is primer combination 1, consisting of primers numbered 1 to 30 in the table below, or primer combination 2, consisting of primers numbered 1 to 20:

[0007]

[0008] .

[0009] Furthermore, primer combination 1 or primer combination 2 is obtained by mixing the primers in equal molar amounts.

[0010] Furthermore, the final concentration of primer combination 1 or primer combination 2 is 50 μM.

[0011] The present invention also provides a reagent comprising the primer combination 1 or primer combination 2 described above.

[0012] The present invention also provides a kit containing primer combination 1 or primer combination 2 as described above, wherein the kit further contains Phi29 DNA polymerase.

[0013] The primer combination 1 or primer combination 2 provided by this invention can be used to amplify the whole genome DNA of sheep embryonic cells or sheep whole genome DNA.

[0014] The reagents provided by this invention are used in amplifying sheep embryonic cell whole genome DNA or sheep whole genome DNA.

[0015] The kit provided by this invention is used for amplifying sheep embryonic cell whole genome DNA or sheep whole genome DNA.

[0016] The beneficial effects of this invention are as follows:

[0017] 1. This invention designs random primers based on sheep reference genome sequences and optimizes the reaction system based on MDA whole genome amplification technology. This enables the amplification of whole genome DNA amplification products with high coverage and low mismatch rate using 10 sheep embryo cells or as low as pg level genomic DNA. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs.

[0018] 2. The primers of this invention are 6-base random primers designed based on the sheep reference genome sequence (ARS-UI_Ramb_v2.0). By ensuring the randomness and uniformity of the primer binding sites in the genome, full-length unbiased amplification can be achieved. The primer length needs to be balanced between random binding ability and specificity, avoiding the occurrence of multiple consecutive identical bases or simple repetitive sequences, so as to reduce non-specific binding and amplification bias. 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.

[0019] 3. The kit of the present invention uses high-fidelity Phi29 DNA polymerase to achieve uniform amplification of the whole genome, with an average amplification product length >10 Kb.

[0020] 4. Using the technical solution of this invention, the three-step operation process (cell lysis - lysis termination - isothermal amplification) can complete efficient amplification within 3.5 hours. Attached Figure Description

[0021] Figure 1 This is an agarose gel electrophoresis image of the whole genome amplification product from Example 1. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments.

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Design and validation of random primers:

[0025] 1.1 Design of random primers: Based on the sheep reference genome sequence (ARS-UI_Ramb_v2.0) (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_016772045.1 / ), 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.

[0026] First, the frequency of 6 bp sequences in the genome was statistically analyzed using the bioinformatics tool Jellyfish, forming a primer library. This library contains 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, resulting in 973 6 bp sequences. Then, the positions of these 973 6 bp sequences in a reference genome were 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 according to predefined parameters.

[0027] The screening parameters are as follows: primer base number is 4; non-self-reverse complementarity; GC content is 50; maximum consecutive base number is <= 2; primers are not reverse complementary; the top 30 primers that meet the above conditions are selected based on their frequency of occurrence.

[0028] Considering that the theoretical amplification length of Phi29 DNA polymerase can reach 70 kb, the primer spacing needs to be much smaller than this. Whole-genome simulation was performed using bioinformatics software to ensure that all regions of the genome can be effectively amplified without significant bias. The primer spacing was determined to be 19 kb.

[0029] After screening, 30 primer sequences were selected. The sequence information of the primers and the simulated genome coverage information of the amplification products are shown in Tables 1 and 2.

[0030] Table 1. Information on 30 random primer sequences

[0031]

[0032]

[0033] Table 2. Simulated information on genome coverage of 30 primer amplification products

[0034]

[0035] 1.2 Verification of random primer amplification effect:

[0036] 1.2.1 Test objective: To confirm the feasibility of using genomic DNA extracted from sheep blood (2ng total input) at a final primer concentration of 50μM; to set up two experimental groups, designated as No. 1 and No. 2, to evaluate the effectiveness of random primers.

[0037] Note: The primer concentrations in the tests below are the sum of the concentrations of each primer in the primer combinations in Table 1.

[0038] 1.2.2 Whole genome amplification: The amplification system is shown in Table 3:

[0039] Table 3. Whole Genome Amplification Reaction System

[0040]

[0041] Note: The phi29 DNA polymerase and 10× phi29 reaction buffer were from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 14404ES80. The random primers were obtained by mixing equimolar amounts of the 30 primers in Table 1.

[0042] Whole genome amplification reaction program: 30℃, 3h, 65℃, 10min, 4℃ incubation. PCR instrument heating cap temperature set to 70℃.

[0043] 1.2.3 Results show that under this experimental system, the primer combinations in Table 1, at a working concentration of 50 μM, can achieve high yields after amplification with only 2 ng of sheep genomic DNA. As shown in Table 4, the concentration of whole genome amplification products in experimental group 1 was 232 ng / μL and that in group 2 was 224 g / μL. Both experimental groups achieved high yields of amplification products, with an average of 9.2 μg.

[0044] Table 4. Concentration of whole genome amplification products

[0045]

[0046] The whole genome amplification products from the two experimental groups were detected using 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, Figure 1 M stands for 10K Marker. The amplification product bands of experimental groups 1 and 2 are around 10kb in size, indicating that the amplified fragments are complete.

[0047] 1.3. Resequencing and library construction of whole-genome amplification products:

[0048] The whole genome amplification products of the samples obtained using the above steps were uniformly used to construct libraries using a DNA resequencing library preparation kit (GenoBaits® DNA Library Prep Kit for Illumina, catalog number GB00010, Borui Biotechnology Co., Ltd.). The library construction method using the DNA resequencing library preparation kit is based on the data generated under the library construction process using the Borui DNA library preparation kit with a DNA input of 200 ng.

[0049] This experiment used sheep genomic DNA products amplified with the primer combinations in Table 1 as described in section 1.2.3 as the experimental group, and used genomic DNA samples extracted from sheep blood as the normal DNA group for comparison.

[0050] The results of resequencing library preparation are shown in Table 5:

[0051] The average coverage of the normal DNA genome resequencing library on the 26 chromosomes was 98.58%. The coverage of the experimental group resequencing library for the whole genome amplified using the primer combination of the present invention was 98.02%, which was not significantly different from the coverage of the normal DNA genome. This indicates that the DNA product after whole genome amplification using the primer combination of the present invention has sufficiently high integrity.

[0052] Table 5. Genome-wide coverage

[0053]

[0054]

[0055] 1.4. Whole-genome amplification product capture and sequencing:

[0056] The amplification products from the experimental group and the normal DNA group obtained in the above steps were uniformly captured using a PHR0072_Oa40K_V3.0 Panel (40767 sites, 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 1.0G to ensure the reliability of the evaluation results. A template-free control group was also included.

[0057] The results are shown in Table 6. The DNA product data amplified by the primer combination of the present invention are comparable to those of the normal DNA group, indicating that the primer combination and amplification system of the present invention are reliable.

[0058] Table 6. Probe capture data of whole genome amplification products

[0059]

[0060] Example 2: Whole genome amplification of sheep embryonic cells:

[0061] This embodiment will use sheep embryo cells to further verify the random primer combinations screened in Example 1.

[0062] To compare the effects of different primer types on amplification, this example used 20 / 30 primer combinations to amplify embryonic cell lysis products.

[0063] The 30 primer combinations are primers numbered 1-30 in Table 1. Each primer was diluted with water to 100 μM according to the synthesis company's requirements, and then mixed evenly in equal volumes to form primer combination 1.

[0064] The 20 primer combinations are primers numbered 1-20 in Table 1. Each primer was diluted with water to 100 μM according to the synthesis company's requirements, and then mixed evenly in equal volumes to form primer combination 2.

[0065] The embryonic cell samples used in this embodiment are all sheep embryonic cells provided by Inner Mongolia Saiqi Company (each embryonic cell sample contains 10 cells).

[0066] Detailed operating procedures:

[0067] 2.1 Cell lysis: Use lysis buffer to lyse cells and release genomic DNA;

[0068] Prepare the lysis buffer according to Table 7:

[0069] Table 7. Lysis Buffer Formulation

[0070]

[0071] Embryonic cells were removed from -80°C, thawed on ice, and briefly centrifuged to collect the liquid at the bottom of the tube. 3 μL of lysis buffer was added to a PCR tube containing 4 μL of cell sample (preserved in calcium- and magnesium-free DPBS, Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500). The tube was gently tapped to mix thoroughly, and the lysis products were briefly centrifuged to collect at the bottom of the tube. The tube was then incubated at 25±2°C for 5 min.

[0072] 2.2 Termination of lysis: Add neutralization buffer to terminate the lysis reaction and prevent DNA degradation.

[0073] Add 3 μL of neutralization solution to the centrifuge tube after step 2.1. The neutralization solution formula is shown in Table 8. Gently tap the tube wall to mix and briefly centrifuge to concentrate the reaction product at the bottom of the tube. Place the sample on ice before preparing the next amplification reaction solution.

[0074] Table 8 Neutralization solution formulation (20 μL)

[0075]

[0076] Note: The solvents for Tris-HCl (400 mM, pH 4.0) and MgCl2 (15 mM) are both nuclease-free water.

[0077] Note: When adding lysis buffer, do not touch the cell surface. Do not use a pipette to blow or agitate the sample, as this may cause cell samples to adhere to the pipette tip. Do not shake vigorously to prevent cell sample loss.

[0078] 2.3 Isothermal amplification: The lysis products obtained in 2.2 were amplified using primer combination 1 with 30 primers and primer combination 2 with 20 primers, respectively.

[0079] The amplification reaction solution system is shown in Table 9:

[0080] Table 9 Amplification reaction solution system

[0081]

[0082] After the amplification reaction solution is mixed well, immediately add 30 μL of the well mixed amplification reaction solution to the sample tube after step 2.2, gently shake to mix, briefly centrifuge to collect the reaction product at the bottom of the tube, and then place it into a PCR instrument with the corresponding program set.

[0083] The PCR instrument was set to 70℃ for heating, and incubated at 30℃ for 3 hours, followed by incubation at 65℃ for 10 minutes to inactivate the phi29 DNA polymerase, and then kept at 4℃.

[0084] 2.4 Amplification product capture and sequencing:

[0085] The whole genome amplification products obtained after amplification in step 2.3 were captured and sequenced according to step 1.4 of Example 1. Six samples were tested using primer combination 1 with 30 primers, and 39 samples were tested using primer combination 2 with 20 primers.

[0086] As shown in Table 10, both primer combinations used in this invention exhibited good amplification uniformity, high capture efficiency, and SNP detection rate in embryonic cells, indicating that this method has reliable applicability in embryonic cell systems.

[0087] Table 10. Probe capture data of sheep embryonic cell whole genome amplification products

[0088]

[0089] Note: All data are averages. The test sample size in the above examples was 10 cells.

[0090] Further comparison of different primer dosages revealed that primer combination 2 performed better than primer combination 1, indicating that the amplification system with 20 primer combinations was superior to that with 30 primer combinations in both amplification uniformity and SNP detection rate, demonstrating that more primer types are not necessarily better. Furthermore, the genotypic concordance rate among different samples from the same embryo exceeded 98.5%, further confirming the high accuracy and reproducibility of this method in SNP genotyping.

[0091] Example 3: Comparative test of primer combinations of the present invention for different cell numbers:

[0092] To compare the amplification differences of the present invention under low and high cell counts, this embodiment will use the 20 primer combinations in Example 2 to test two samples (10 cells and >100 cells) from the same embryo.

[0093] Six embryonic cell samples were tested in this experiment, each with a volume of 4 μL. The embryonic cells were stored in calcium and magnesium-free DPBS (Shanghai Xiaopeng Biotechnology Co., Ltd., catalog number: C3590-0500). The cells were treated with lysis buffer and neutralization solution, and the resulting cell lysis products were amplified. The amplified products were then captured and sequenced, following the same procedures as in Example 2.

[0094] The results are shown in Table 11.

[0095] Table 11. Probe capture data of whole-genome amplification products with different cell numbers

[0096]

[0097] Test data show that primer combination 2 of the present invention does not show a significant difference in performance between 10 cells and more than 100 cells, and even performs better in a few cell groups. This indicates that when using the primer combination of the present invention to amplify the whole genome of sheep embryonic cells, the data quality does not depend entirely on the number of cells. As long as the quality of the cells themselves is high enough, excellent data results can still be obtained even if the starting cell number is low.

[0098] In summary, the primer combination of the present invention can rapidly and efficiently amplify whole-genome DNA amplification products with high coverage and low mismatch rate using only 10 sheep embryo cells or as low as pg of genomic DNA. It can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs.

[0099] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0100] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A primer combination for amplifying the whole genome DNA of sheep embryonic cells, characterized in that, The primer combination is primer combination 1 consisting of primers numbered 1 to 30 in the table below, or primer combination 2 consisting of primers numbered 1 to 20: 。 2. The primer combination for amplifying the whole genome DNA of sheep embryonic cells according to claim 1, characterized in that, The primer combination 1 or primer combination 2 is obtained by mixing the primers in equal molar amounts.

3. The primer combination for amplifying the whole genome DNA of sheep embryonic cells according to claim 2, characterized in that, The final concentration of primer combination 1 or primer combination 2 is 50 μM.

4. A reagent comprising primer combination 1 or primer combination 2 as described in claim 1.

5. A kit comprising primer combination 1 or primer combination 2 as described in claim 1, characterized in that: The kit also contains Phi29 DNA polymerase.

6. The use of primer combination 1 or primer combination 2 as described in claim 1 in amplifying sheep embryonic cell whole genome DNA or sheep whole genome DNA.

7. The use of the reagent according to claim 4 in amplifying sheep embryonic cell whole genome DNA or sheep whole genome DNA.

8. The use of the kit according to claim 5 in amplifying sheep embryonic cell whole genome DNA or sheep whole genome DNA.

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