A primer set, kit and method for quality control of whole genome amplification of cynomolgus monkey

Abstract

The application belongs to the technical field of biology, and discloses a primer group, a kit and a method for quality control of whole genome amplification of Macaca fascicularis. The quality control method for the whole genome amplification product of Macaca fascicularis is simple and easy to operate, 6 pairs of primers can be used in the same reaction tube, thereby reducing the steps of adding reagent components and sub-packaging, and the whole reaction has low requirements on laboratory conditions, only a conventional PCR amplification laboratory, agarose electrophoresis and a gel analyzer are needed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a primer set, a kit and a method for quality control of whole genome amplification of Macaca fascicularis. BACKGROUND

[0002] Whole genome amplification (WGA) is a technology for non-selective amplification of all genomic sequences. The main purpose is to increase the amount of DNA to the maximum on the basis of reflecting the whole genome as it is, and to amplify the entire genomic DNA of trace tissue or single cell without sequence bias, so as to provide sufficient DNA template for subsequent multi-gene, multi-site analysis and comprehensive genomic research. At present, WGA is widely used in oncology, neurology and developmental biology, and can solve the problem of insufficient DNA sample amount.

[0003] According to the different principles, WGA technology is mainly divided into two types: one is based on thermal cycling and PCR-based amplification technology, such as degenerate oligonucleotide primer PCR (DOP-PCR), ligation-mediated PCR (LM-PCR), primer extension reaction before amplification (PEP) and multiple annealing loop-based amplification of circles (MALBAC) and the like; the other is based on isothermal reaction and non-PCR-based amplification technology, such as multiple displacement amplification (MDA) and primerase-based whole genome amplification (pWGA). At present, the methods of MDA and MALBAC are widely used in clinic with good amplification efficiency, genomic coverage and amplification uniformity. These two WGA methods have their own advantages and disadvantages: MDA can amplify longer DNA fragments (more than 2000 bp), but the uniformity of amplified genome is not ideal, that is, there is a problem of exponential amplification of DNA in some regions. The MDA amplification product is suitable for the diagnosis of point mutations and large fragment deletions and duplications based on PCR (GAP-PCR) and gene chip (SNP haplotype analysis) and the like, but the detection effect of genomic copy number variation is poor; the MALBAC amplification product can uniformly cover the whole genome, but the product fragment length is short (about 300-700 bp), which is suitable for the analysis of point mutations and chromosomal copy number variations based on PCR and NGS and the like.

[0004] With the continuous development of life science, Macaca fascicularis as an experimental animal is paid more and more attention. At the same time, the sample of Macaca fascicularis, especially the embryonic sample, is difficult to obtain, and WGA can provide sufficient DNA sample amount for downstream experiments, but there is currently a lack of quality control method for WGA product of Macaca fascicularis. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide an efficient, simple and reliable method for evaluating the quality of cynomolgus monkey WGA products, which can effectively improve the success rate and quality of downstream experiments and reduce experimental costs.

[0006] The primary purpose of the present application is to provide a primer set for quality control of cynomolgus monkey whole genome amplification.

[0007] The second purpose of the present application is to provide a kit containing the above-mentioned primer set.

[0008] The third purpose of the present application is to provide a method for quality control using the above-mentioned primer set.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] A primer set for quality control of cynomolgus monkey whole genome amplification, comprising the primer sequences shown in SEQ ID NO: 1-SEQ ID NO: 12.

[0011] The present application selects PGK1, RPS19, GAPDH, RPL32, ACTB and MRFAP1 as six housekeeping genes for quality control of cynomolgus monkey whole genome, designs primers for each of the six housekeeping genes and performs screening, since the PCR reaction is performed in one tube, in order to amplify the six housekeeping genes at one time, and the different bands can be clearly distinguished in the gel electrophoresis map, the primer combination with a size difference of >40bp between the products is selected. Finally, the above-mentioned primer set of the present application is obtained.

[0012] The present application also provides a kit containing the primer set.

[0013] The present application also provides the use of the kit for quality control of cynomolgus monkey whole genome amplification.

[0014] The present application also provides a method for quality control of cynomolgus monkey whole genome amplification, which uses the primer set to perform PCR amplification of cynomolgus monkey whole genome.

[0015] Preferably, in the PCR amplification, the concentration of each primer in the primer set is 0.004μmol.

[0016] Preferably, the enzyme used in the PCR amplification is Taq Pro Multiplex DNA Polymerase multiplex PCR enzyme.

[0017] Preferably, in the above-mentioned method, the PCR amplification result determination principle is: agarose gel electrophoresis is used, if more than 5 products are amplified, it indicates that the quality of the cynomolgus monkey whole genome used is good, otherwise, it indicates that the quality of the cynomolgus monkey whole genome used is not good.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] 1. The application provides a quality control method for Cynomolgus monkey whole genome amplification products, which improves the success rate and quality of downstream experiments; 2. The quality control method for Cynomolgus monkey whole genome amplification products is simple and easy to operate, 6 pairs of primers can be amplified in the same reaction tube, thereby reducing the steps of adding reagent components and sub-packaging, and the whole reaction has low requirements on laboratory conditions, only a conventional PCR amplification laboratory, agarose electrophoresis and a gel analyzer are needed. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a gel electrophoresis map of the housekeeping gene primer amplification product;

[0021] Figure 2 is a Cynomolgus monkey WGA product, which is amplified by 6 pairs of primers, mixed with 6 kinds of single amplification products, and the agarose gel electrophoresis result map after amplification of the application, PGK1, RPS19, GAPDH, RPL32, ACTB and MRFAP1 are the amplification results of the 6 pairs of primers, Mix is the mixed result of the 6 kinds of single amplification products, and multiplex PCR is the amplification result of the application;

[0022] Figure 3 is the amplification result of the application on 40 Cynomolgus monkey embryo WGA products. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical scheme and advantages of the application, the application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents and the like used can be obtained from commercial channels unless otherwise specified.

[0024] Example 1 primer screening

[0025] Primers (such as Tables 1 to 6) are designed for 6 housekeeping genes MRFAP1, ACTB, RPL32, GAPDH, RPS19 and PGK1, and then PCR amplification is carried out by using the designed primers, and the PCR reaction system of each housekeeping gene is as follows: Cynomolgus monkey DNA 50-200 ng, upstream primer 2 μl, downstream primer 2 μl, 2×Hieff TM PCR Master Mix 25 μl, ddH2O up to 50 μl, and the PCR amplification conditions are as follows: 95℃, 5 min, 1 cycle, 95℃, 30 s, 60℃, 30 s, 35 cycles, 72℃, 30 s, 72℃, 10 min, 1 cycle.

[0026] Table 1 MRFAP1 primer

[0027]

[0028] Table 2 ACTB Primers

[0029]

[0030] Table 3 RPL32 primers

[0031]

[0032] Table 4 RPS19 primers

[0033]

[0034] Table 5 GAPDH Primers

[0035]

[0036] Table 6 PGK1 primers

[0037]

[0038] The gel electrophoresis images of the amplification products of the above primers are shown below. Figure 1 The primers that can amplify specific bands of the correct size include: MRFAP1-2, MRFAP1-4, ACTB-3, RPL32-1, RPL32-2, RPL32-4, RPS19-1, RPS19-2, RPS19-3, RPS19-4, GAPDH-2, GAPDH-3, GAPDH-4, PGK1-2, PGK1-4, PGK1-5, and PGK1-6.

[0039] To amplify six housekeeping genes at once and clearly distinguish different bands in the gel electrophoresis image, primer combinations with a size difference of >40bp between products were selected. The final primers used were MRFAP1-4, ACTB-3, RPL32-2, GAPDH-2, RPS19-1, and PGK1-6.

[0040] Example 2: Quality control method for cynomolgus monkey whole genome amplification products

[0041] After the six primer pairs obtained in Example 1 were synthesized, they were purified by PAGE and lyophilized into powder for storage and transportation; the six primer pairs and the fragment lengths of the amplification products are shown in Table 7.

[0042] Table 7

[0043]

[0044] 6 pairs of primers are diluted with ddH2O to 10 μmol / L of mother liquor, respectively, then 20 μl of the upstream and downstream primer mother liquor are mixed together and adjusted to 0.2 μmol / L to prepare the primer working solution; 50-100 ng of WGA product and 6 μl of the primer working solution are taken to perform PCR reaction by using Taq Pro Multiplex DNA Polymerase or other high-efficiency and high-fidelity polymerase; after the PCR reaction, 10 μl of the product is taken and mixed with 2 μl of 6×DNA loading buffer, and then electrophoresis is performed on a 2% agarose gel at 120 V for 30 min, and then ultraviolet light imaging is performed on a gel imaging instrument; if the template DNA quality is good, 6 products can be theoretically amplified; if there are 5 or more amplification products, it is indicated that the template DNA can be used for downstream detection, and the more and clearer the bands are, the better the quality of the template DNA is.

[0045] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for quality control of whole-genome amplification in cynomolgus monkeys, characterized in that, Multiplex PCR amplification was performed using the primer sequences shown in SEQ ID NO: 1 to SEQ ID NO:

12. The result was judged as follows: if more than 5 products were amplified by agarose gel electrophoresis, it indicated that the quality of the cynomolgus monkey whole genome was good; otherwise, it indicated that the quality of the cynomolgus monkey whole genome was poor.

2. The method according to claim 1, characterized in that, In PCR amplification, the concentration of each primer in the primer set is 0.004 μmol.

3. The method according to claim 1, characterized in that, The enzyme used for PCR amplification is Taq Pro Multiplex DNA Polymerase.

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