KR102768600B1 - Primer Pair having Miso-Winglet Structure for Allele Analysis, and Method for Analyzing Alleles of SNP using the Same - Google Patents

Abstract

The present invention was designed to solve the problem of difficulty in analyzing alleles of multiple single nucleotide sequence variations with a single multiplex polymerase chain reaction (multiplex-PCR), and is a method for detecting each allele of one or more single nucleotide sequence variations based on a multiplex polymerase chain reaction (multiplex-PCR) using an oligonucleotide of a Miso-Winglet structure.

Description

{Primer Pair having Miso-Winglet Structure for Allele Analysis, and Method for Analyzing Alleles of SNP using the Same}

The present invention was devised to solve the problem of difficulty in applying a multiplex polymerase chain reaction (multiplex-PCR) method to a large number of single nucleotide polymorphisms when analyzing all alleles of a single nucleotide polymorphism by a conventional PCR method, and relates to an oligonucleotide having a Miso-Winglet structure and a method for detecting each allele of one or more single nucleotide polymorphisms (SNPs) by a multiplex polymerase chain reaction (multiplex-PCR) using the same.

Single nucleotide variant (SNV) refers to a mutation in the genome of an organism that exhibits a difference in a single nucleotide sequence, and includes single nucleotide polymorphism (SNP) and point mutation.

Single nucleotide polymorphisms (SNPs) refer to a change in a specific base sequence at the same location in the genome of the same species, which results in a different trait being expressed, while point mutations occur when a single base sequence is substituted, inserted, or deleted, and can block or alter the production of a specific protein.

These single nucleotide sequence variations are attracting attention because they can be used to understand the genetic function of diseases, and can be used to develop personalized medicine for the prevention or treatment of diseases, and can also be used to distinguish between genetically distinct individuals.

Various detection methods using PCR technology are being used to detect single nucleotide sequence mutations. Representative examples include the real-time PCR method that uses a fluorescent substance on a DNA probe to detect single nucleotide sequence mutations, and the AS-PCR (Allele-specific PCR) method that uses the single nucleotide sequence mutation location at the 3' end of the primer.

Real-time PCR methods using fluorescent substances have the advantage of being able to analyze results in real time and eliminating the need for follow-up analysis experiments. However, real-time PCR methods using fluorescent substances have a limitation of using only five channels for fluorescence of a specific wavelength range, so if only one of the two alleles of a single nucleotide sequence variation is analyzed, multiplexing of five single nucleotide sequence variations is possible, but only one or two single nucleotide sequence variations can be analyzed to confirm both alleles, and there are disadvantages in that a lot of costs are incurred for using fluorescent substances and real-time PCR equipment.

The conventional PCR (general PCR) analysis method using AS-PCR (Allele-specific PCR) technology has the advantage of being able to analyze more than 10 single nucleotide sequence mutations by applying multiplex-PCR technology when only one of the two alleles of a single nucleotide sequence mutation is analyzed. However, in order to confirm both alleles of a single nucleotide sequence mutation, ARMS-PCR (Amplification refractory mutation system) technology using four primers must be applied.

ARMS-PCR technology is a technology designed to produce specific primers for two different alleles in a double-stranded DNA to be used as a template strand, so that they can bind in different directions.

This ARMS-PCR technique is useful for distinguishing a single nucleotide sequence mutation, but since additional amplification products are generated in addition to the amplification products for each allele, when applying the multiple amplification technique to two or more single nucleotide sequence mutations, the number of amplification products increases by a multiple of three, which increases the possibility of generating non-specific amplification products, and it is difficult to group and list the sizes of amplification products by amplification product for each single nucleotide sequence mutation when designing primers.

To solve these difficulties, the present invention invented a method for designing PCR primers that control the size of the amplification product while designing two alleles of a single nucleotide sequence mutation unidirectionally and do not generate additional amplification products other than the amplification products of each allele.

This technology has the advantage of being able to simultaneously identify multiple alleles of single nucleotide sequence mutations in a single experiment by applying multiplex amplification technology (multiplex-PCR).

The purpose of the present invention is to design PCR primers for each allele in the same direction based on the sequence of a template strand to be amplified when analyzing each allele of a single nucleotide sequence mutation by a PCR method, the design method using one complementary forward or reverse primer of the template strand and two complementary forward or reverse primers of each allele.

More specifically, when designing PCR primers for each allele of a single nucleotide sequence mutation, the size of the amplified product of each allele is controlled by adding a sequence complementary to the template strand to the 5' end, and by replacing a specific part of the overlapping sequence of another allele with a different base to compete for the hybridization process between the PCR primers of each allele to the template strand, thereby increasing the specificity of the PCR primers.

In order to achieve the above purpose of the present invention,

The present invention provides a primer pair for allele analysis, characterized in that it consists of (A) a primer-1 for allele-1, which includes a terminal base X of a mutation site of an allele and has a size of 10 to 30 bases in the 5'-direction, and a front region of a sufficient size to be distinguished from a PCR product by primer-2 in the 5'-direction of the rear region, and has a mutation part A in which 1 to 3 consecutive bases in the 5'-direction of the rear region are non-complementary to a template, and the remaining bases are complementary to the bases of allele-1; and (B) a primer-2 for allele-2, which includes a terminal base X' of a mutation site of an allele and has a size of 10 to 30 bases in the 5'-direction and is complementary to a template.

In the present invention, 1 to 3 consecutive bases of a non-complementary mutation B and/or mutation C, C' can be added to the rear region of the primer-1 or 1 or 2 positions of the primer-2.

In the present invention, the structure of the primer pair according to the present invention as described above is referred to as the 'Miso-Winglet structure'.

In accordance with the above purpose, the present invention provides a method for analyzing alleles, which includes a step of detecting allelic differences of single nucleotide sequence variations by polymerase chain reaction (PCR) using a primer pair of a Miso-Winglet structure. In the case of multiple single nucleotide sequence variations, it may also be possible to analyze alleles of multiple single nucleotide sequence variations by simultaneously using primer pairs for each allele.

Such an analysis method may include: (a) a step of preparing a nucleic acid sample isolated from a sample of an individual; (b) a step of performing a multiplex polymerase chain reaction (multiplex-PCR) using a primer pair of a Miso-Winglet structure with the isolated nucleic acid sample as a template strand; and (c) a step of analyzing a single nucleotide polymorphism.

In addition, the present invention provides a method for diagnosing the origin of a biological product by using the method of analyzing the aforementioned allele.

At this time, the biological product is food, and the origin may be one or more including a breed, a place of origin, a producer, and a producing individual. For example, as presented in the example below, the biological product is milk, and the origin may be a breed of cattle that produced the milk, which may be utilized to determine the species origin of the milk.

Additionally, the biological product may be a pathogenic organism and may be derived from a genotype of the pathogenic organism.

In addition, the present invention provides a method for predicting future characteristics of crops or livestock by using the method for analyzing the aforementioned alleles. For example, as presented in the examples below, a method for predicting beef yield and quality of cattle can be provided by analyzing alleles of various single nucleotide sequence variations in beef cattle.

The present invention also provides a PCR reaction composition or an allele analysis kit comprising a primer pair for allele analysis as described above.

The amplification of the present invention is performed according to PCR. Primers are used in the gene amplification reaction.

The 'target amplification product' of the present invention refers to an amplified nucleic acid (DNA) sequence for a target to be detected, and is a product produced by polymerase chain reaction (PCR). The 'target amplification product' is no different from the term 'amplification product' used in this specification, and they are used interchangeably in this specification.

The term 'primer' used in the present invention means a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and functions as a starting point for copying the template strand. The appropriate length of the primer may vary depending on the intended use, but may generally consist of 15 to 30 bases. The primer sequence does not need to be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. These nucleic acid sequences can also be modified using many methods known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologues of a natural nucleotide, and modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

When the method of the present invention is performed using a primer, multiplex polymerase chain reaction (multiplex PCR) can be performed to simultaneously detect target genes in the analysis subject. Therefore, the method of the present invention performs a gene amplification reaction using a primer that binds to DNA extracted from a sample.

In one embodiment of the present invention, the Multiplex PCR reaction conditions may be performed once at 95°C for 15 minutes, followed by 35 cycles of 95°C for 20 seconds, 60°C for 40 seconds, and 72°C for 60 seconds, and then performed once at 72°C for 3 minutes.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these examples.

The present invention provides a primer pair of a Miso-Winglet structure that can prevent the generation of unnecessary amplification products, which is a problem in analysis using ARMS-PCR (Amplification refractory mutation system) in single nucleotide polymorphism, and can generate only two target amplification products, and can simultaneously analyze a large number of single nucleotide polymorphism alleles by multiplex polymerase chain reaction, which was impossible with existing analysis methods, and therefore can be usefully used in various PCR diagnostic and assay methods.

Figure 1 is a schematic diagram of a PCR primer pair using an oligonucleotide of the Miso-Winglet structure of the present invention.
Figure 2a is a schematic diagram showing whether the primers of each allele bind to the template strand and are amplified during the PCR process using the primer pairs exemplified in Figure 1.
Figure 2b is a schematic diagram showing the binding and amplification relationship between the primers of each allele and the amplified product template strand when the amplified product generated during the PCR process of the Miso-Winglet PCR primer used in Figure 1 is used as the template strand.
Figures 3 to 6 are photographs of the electrophoresis results of Multiplex PCR products in an embodiment of the present invention.

Hereinafter, preferred embodiments are presented to help understand the present invention, but the following embodiments are only illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and technical idea of the present invention, and it is natural that such changes and modifications fall within the scope of the appended patent claims.

As described above, the invention relates to a primer pair for allele analysis, characterized by comprising: (A) a primer-1 for allele-1, which includes a terminal base X of a mutation site of an allele and has a size of 10 to 30 bases in the 5'-direction, and a front region of a size sufficient to be distinguished from a PCR product by primer-2 in the 5'-direction of the rear region, and has a mutation part A of 1 to 3 consecutive bases in the 5'-direction of the rear region that is non-complementary to a template, and the remaining bases are complementary to the bases of allele-1; and (B) a primer-2 for allele-2, which includes a terminal base X' of a mutation site of an allele and has a size of 10 to 30 bases in the 5'-direction and is complementary to a template. An example of such a primer pair is illustrated in Fig. 1(a) as a schematic diagram of a primer pair of a Miso-Winglet structure consisting of primer-1 of allele type A and primer-2 of allele type G. In the drawing, the mutation A of primer-1, indicated in bold, is an artificial mutation, and the corresponding region A' of primer-2 is indicated in bold to make it easy to see the same position, and this part is not a mutation.

The primer pairs in Tables 1 and 3 are distinguished by underlining the shear region, and each mutation and terminal base are indicated in the second row of the table.

In Tables 1 and 3 below, sequence numbers 1, 2, 3, 7, 10, 13, 16, and 19 correspond to primer-1, and sequence numbers 4, 5, 8, 11, 14, 17, and 20 correspond to primer-2. The underlined part in primer-1 is the front end region and the rest is the rear end region, and non-complementary bases, that is, mutation regions, such as variant parts B and/or variant parts C, C', can be added between the rear end region and both ends of primer-2 (corresponding part B' is not a mutation). An example of such a primer pair is illustrated in Fig. 1 (b), in which the front end region, the rear end region, variant parts A to C, corresponding parts A', B', variant part C', and terminal bases X, X' are exemplarily indicated. The mutations A to C of primer-1 and mutation C' of primer-2, which are indicated in bold in the drawing, are artificial mutations, and the corresponding regions A' and B' of primer-2 are indicated in bold to make it easier to see the same location, and these parts are not mutations.

In Tables 1 and 3 below, sequence numbers 6, 9, 12, 15, 18, and 21 are reverse primers that commonly correspond to the forward primers, Primer-1 and Primer-2. In the following, unless otherwise specified, it is assumed that reverse common primers are necessarily used together for PCR. To avoid confusion, the following explanations are given in order by numbering them.

(1) Shear area of primer-1

The shear region of primer-1 for the above allele-1 is a region capable of distinguishing the sizes of allele-2 or additional PCR amplification products, and has an advantage in that it is useful for designing amplification products at specific intervals or desired sizes when distinguishing two or more amplification products in multiplex polymerase chain reaction (Multiplex-PCR) through agarose gel electrophoresis or automatic electrophoresis device analysis. At this time, the size of the shear region may be 10 to 50 bases, but is not limited thereto. However, if it is too large, it is a waste of resources, and if it is too small, it becomes difficult to distinguish PCR products. An average person of ordinary skill in the art to which the present invention belongs will be able to determine an appropriate size depending on the situation.

(2) Mutant A

If there is no sequence difference between the corresponding 5' terminal portions of primer-1 and primer-2, when the amplified product using primer-1 or primer-2 is used as the template strand during the PCR process, the binding specificity between the template strand of the amplified product using the PCR primer of each allele and the PCR primer of each allele may decrease, which may result in inaccurate PCR results. Therefore, by forming a mutant portion A, which is an artificial mutant nucleotide sequence, at the 5' terminal portion of the primer-1 terminal portion, when the template strand and the PCR primer of each allele bind, there is an advantage of suppressing the biased binding of the allele-1 PCR primer when the template strand and the PCR primer of each allele bind, and suppressing the amplified product of allele-2 using the allele-2 PCR primer during the PCR process from being used as the template strand of the allele-1 PCR primer.

(3) Terminal base X, X'

The 3' terminal portions of primer-1 and primer-2 are the allelic positions of the single nucleotide sequence mutation and are complementary to the corresponding bases of the template. Therefore, the 3' terminal sequence X of primer-1 and the 3' terminal sequence X' of primer-2 are different bases.

(4) Variant B (optional)

In the PCR process using primer-1 and primer-2 according to the present invention, when the amplified product by primer-1 or primer-2 is used as the template strand, the binding specificity between the template strand of the amplified product using the PCR primer of each allele and the PCR primer of each allele may decrease, thereby obtaining inaccurate PCR results. To solve this problem, in the present invention, a mutation B in which 1 to 3 consecutive bases are additionally substituted so as to be non-complementary to the template may be provided at 1 or 2 locations in the rear region of primer-1. By doing so, ⓐ primer-1 can inhibit binding to the template strand of the amplified product using primer-2 during the PCR process due to a difference in Tm value due to a difference in sequence, or ⓑ primer-2 can inhibit binding to the template strand of the amplified product using primer-1 during the PCR process due to a difference in Tm value.

(5) Mutant C, C' (optional)

In the present invention, in order to increase the specificity of the sequence difference between the 3'-terminal bases X and X' of primer-1 and primer-2 corresponding to the allelic position of the single nucleotide sequence mutation, non-complementary mutation parts C and C' can be placed at 1 to 2 bases at the 2nd to 4th positions from the 3' end. At this time, the mutation parts C and C' of primer-1 and primer-2 are composed of different bases.

The mutations C and C' of primer-1 and primer-2 are regions that increase the specificity of the allele position, and serve to increase the specificity of the PCR primer for each allele by using a sequence that is not complementary to the template strand.

In the present invention as described above, it is preferable that each mutation portion has 1 to 3 mutations in order to change the binding specificity of each primer. If more than 3 mutations are included, the binding force between the primer and the template strand is greatly reduced, which adversely affects PCR efficiency.

FIG. 2a is a schematic diagram showing the binding and amplification of the primers of each allele and the template strand during the PCR process using the primer pairs exemplified in FIG. 1, and FIG. 2b is a schematic diagram showing the binding and amplification relationship between the primers of each allele and the template strand of the amplified product when the amplified product generated during the PCR process of the Miso-Winglet PCR primers used in FIG. 1 is used as the template strand.

In Fig. 2a, (a) and (a`) represent the binding and amplification of each allele PCR primer when the genotype of the template strand is A, and in Fig. 2a, (b) and (b`) represent the binding and amplification of each allele PCR primer when the genotype of the template strand is G.

In Figure 2b, (a) represents the process in which the PCR primer of allele A type binds to the template strand of gDNA and is amplified, and (a`) represents the binding and amplification of the primer of each allele when the amplification product created in (a) is used as the template strand.

In Figure 2b, (b) represents the process in which the PCR primer of the allele G type binds to the template strand of gDNA and is amplified, and (b`) represents the binding and amplification of the primer of each allele when the amplification product created in (b) is used as the template strand.

The structure of the primer pair oligonucleotide according to the present invention as described above is first proposed by the present inventors, and is named the 'Miso-Winglet' structure after being inspired by the winglet shape of a small wing attached vertically or nearly vertically to the tip of an airplane's main wing, and a PCR primer having this structure is named the 'Miso-Winglet primer'.

When the above Miso-Winglet structure oligonucleotide is used as an allele PCR primer for a single nucleotide sequence variation, the hybridization specificity of each allele PCR primer in a multiplex polymerase chain reaction (multiplex-PCR) is improved, and the size of the PCR amplification product can be freely controlled, so it can be usefully used to detect a number of single nucleotide sequence variations simultaneously.

More specifically, the Miso-Winglet primer is a new concept structure primer developed by the present inventor, which can design a PCR primer for each allele of a single nucleotide sequence mutation in the same direction as the template strand of a sample nucleic acid, and can control the size of the target amplification product of each allele by adding a shear region only to the primer for one allele, thereby enabling more accurate result analysis, and including an artificially mutated nucleotide in a specific region of the two primers (A; B and/or C, C' can be added) and a nucleotide showing a difference between the alleles in the X, X' region, the primer binding specificity for the template strand of the sample nucleic acid or the template strand of the amplification product using the PCR primer of each allele can be greatly improved.

[Example]

Example 1

(1) Materials

Jersey raw milk (Seoul Milk Research Institute), commercially distributed Holstein milk, Simple-Step 2X multiplex PCR Master Mix (Misogene), Oligonucleotide (Cosmogenetech), 50 ~ 500 mM Tris-HCl (pH 9.0), 1 ~ 10 mM MgCl2, 10 ~ 500 mM (NH4)2SO4, 0.001 ~ 0.1% BSA, Hot start Taq DNA polymerase (2.5 U/㎕), Genomic DNA Prep Kit (Nanohelix), HPLC water (Merck)

(2) DNA preparation

DNA extraction was performed using Genomic DNA Prep Kit (Cat No. GCTN200) from Nanohelix. DNA quantitative analysis was performed using Spectrophotometer ND-2000 (Nanodrop, USA), and the concentration and purity of DNA were confirmed by measuring the absorbance at 260 to 280 nM. The extracted DNA was adjusted to 20 ng/㎕ using sterile distilled water and stored at -20℃ until experimental use.

(3) Design of Miso-Winglet primers for single nucleotide sequence mutation PCR analysis

In order to amplify the DNA extracted in (2) above, primers were designed to recognize the bovine MC1R (Bostaurus melanocortin 1 receptor, AF445642.1) gene registered in NCBI.

In more detail, in order to analyze the single nucleotide polymorphism alleles C and T of the codon encoding the 99th amino acid of the MC1R gene of cattle, the corresponding primers for alleles C and T were set in the same direction, and the Miso-Winglet structure was utilized to design the allele C primer (Table 1, SNP Type C Miso-Winglet F Primer).

More specifically, when alleles C and T are set in the same direction, a 50-base oligonucleotide sequence (transfer region) was added to the allele C primer (SEQ ID NO: 1) so that they can be distinguished by the size difference of each amplification product, and artificial mutations having a different sequence from the allele T primer (SEQ ID NO: 4) were provided in the A, B, C, and X regions so that interference between each primer could be minimized.

The above primer sequences are represented by SEQ ID NOs. 1 to 6. Primers for the entire base sequence of SEQ ID NOs. 1 to 6 were synthesized (Cosmogene Tech Co., Ltd.). Information on each primer and the size of the amplified product, etc. are presented in detail in [Table 1] below.

Sequence numbers 1 and 3 correspond to primer-1 in the present invention, and sequence number 2 is a comparative primer produced to be used as a comparative example to confirm the effect of primer-1. As can be seen in the table below, mutation parts A and B of sequence number 2 are composed of the same bases as the corresponding parts A' and B' of primer-2 (sequence number 4).

(4) Setting Multiplex PCR conditions

The PCR reaction solution was prepared by mixing 1 ㎕ (20 ng/㎕) of the genomic DNA extracted from each milk in the above Example (a), 10 ㎕ of Simple Step 2X Multiplex PCR Master Mix, and 3 ㎕ of the mixed primer mixture, and then adding sterilized distilled water to make a final volume of 20 ㎕. The optimized PCR reaction temperature and time were determined to be 95 °C for 15 minutes once, 35 cycles of 95 °C for 20 seconds, 60 °C for 40 seconds, and 72 °C for 60 seconds, and then 72 °C for 3 minutes once. To confirm the final reaction product, 5 ㎕ of the PCR reaction product was loaded onto a 3% agarose gel, electrophoresis was performed at 250 V for 30 minutes, and the results were confirmed with a UV trans-illuminator.

(5) Step of analyzing single nucleotide polymorphism; Confirmation of milk discrimination

Multiplex PCR was performed under the conditions (4) above for two types of Jersey milk and two types of Holstein milk, and the electrophoresis results are shown in Figures 3 and 4.

① Comparative example: Primer containing only the shear, C, and X regions of the Miso-Winglet structure

As can be seen in Fig. 3, the results of Multiplex PCR using the mixed primers of sequence numbers 2, 4, and 6 from the primer information in Table 1 above showed that a 250-bp PCR amplification product, which is an amplification product of the MC1R 296C allele, was confirmed in Holstein milk (Fig. 3, numbers 1 and 3), but a 200-bp PCR amplification product, which is an amplification product of the MC1R 296T allele, was not confirmed in Jersey milk (Fig. 3, numbers 2 and 4).

More specifically, the primer (SEQ ID NO: 2) of the allele C type in the above Example (b) Table 1 did not apply the entire region of the Miso-Winglet structure, but added a 50-base oligonucleotide front region that can distinguish the size of the amplification product; the C and X regions have sequence differences from the primer (SEQ ID NO: 4) of the allele T type, but the A and B regions have no sequence differences.

Therefore, the primer of allele C type (SEQ ID NO: 2), which has no sequence of artificial mutations in the A and B regions, had a higher primer binding affinity with the template strand than the primer of allele T type (SEQ ID NO: 4), which induced biased amplification of the primer of allele C type, and as a result, PCR amplification products were confirmed only in Holstein milk of allele C type, and no PCR amplification products were confirmed in Jersey milk of allele T type, which was inferior in primer binding competitiveness.

② Application example: Primer containing only the shear, A, and X regions of the Miso-Winglet structure (application of primer pair according to claim 1)

As can be seen in Fig. 4, as a result of Multiplex PCR using the mixed primers of sequence numbers 3, 5, and 6 from the primer information in Table 1 above, a 250 bp PCR amplification product, which is an amplification product of the MC1R 296C allele, was confirmed in Holstein milk (Fig. 4, numbers 1 and 2), and a 200 bp PCR amplification product, which is an amplification product of the MC1R 296T allele, was confirmed in Jersey milk (Fig. 4, numbers 3 and 4).

More specifically, the primer (SEQ ID NO: 3) of the allele C type in Table 1 of the above Example 1 did not apply the entire region of the Miso-Winglet structure, but added a 50-base oligonucleotide front region that can distinguish the size of the amplification product; the A and X regions have sequence differences from the primer (SEQ ID NO: 3) of the allele T type, but the B and C regions have no sequence differences.

The primer of allele C type (SEQ ID NO: 3) had a higher primer binding affinity to the template strand than the primer of allele T type (SEQ ID NO: 5), which caused biased amplification of the primer of allele C type. However, even if only the A and X regions of the Miso-Winglet structure were used, PCR amplification products of each allele could be confirmed.

③ Application example: Primer using all areas of the Miso-Winglet structure (application of primer pair according to claim 2)

As can be seen in Fig. 5, the results of Multiplex PCR using the mixed primers of sequence numbers 1, 4, and 6 from the primer information in Table 1 above, a 250 bp PCR amplification product, which is an amplification product of the MC1R 296C allele, was confirmed in Holstein milk (Fig. 5, numbers 1 and 2), and a 200 bp PCR amplification product, which is an amplification product of the MC1R 296T allele, was confirmed in Jersey milk (Fig. 5, numbers 3 and 4).

In the above application example ②, a somewhat biased amplification was observed in the amplification product of allele C type, but in the Multiplex PCR results using primer-1 (sequence number 1) of the Miso-Winglet structure having an additional artificial mutation sequence, the amplification products of each allele were uniformly amplified.

From the above results, it can be confirmed that the Multiplex PCR result using the primer of the Miso-Winglet structure according to the present invention suppresses the interference effect and biased amplification between primers when the primer of the allele of the single nucleotide polymorphism is designed unidirectionally, and thus, unlike the existing ARMS-PCR (Amplification refractory mutation system) technology that uses four primers, it has the great advantage of not generating unnecessary amplification products by using three primers and generating only the target amplification products of each allele.

Example 2

(1) Materials

Korean beef, imported beef, Simple-Step 2X multiplex PCR Master Mix (Misogene), Oligonucleotide (Cosmogenetech), 50 ~ 500 mM Tris-HCl (pH 9.0), 1 ~ 10 mM MgCl2, 10 ~ 500 mM (NH4)2SO4, 0.001 ~ 0.1% BSA, Hot start Taq DNA polymerase (2.5 U/㎕), Genomic DNA Prep Kit (Nanohelix), HPLC water (Merck)

(2) DNA preparation

DNA extraction was performed using Genomic DNA Prep Kit (Cat No. GCTN200) from Nanohelix. DNA quantitative analysis was performed using Spectrophotometer ND-2000 (Nanodrop, USA), and the concentration and purity of DNA were confirmed by measuring the absorbance at 260 to 280 nM. The extracted DNA was adjusted to 20 ng/㎕ using sterile distilled water and stored at -20℃ until experimental use.

(3) Design of Miso-Winglet primers for analyzing 5 single nucleotide sequence mutations (10 alleles in total) by multiplex PCR

To amplify the DNA extracted in the above example (a), the bovine DNA registered in NCBI

PLAG1 (PLAG1 zinc finger,

Primers were designed to recognize the SCD (stearoyl-CoA desaturase, AY241932.1) gene.

The polymorphisms of each gene reflect the characteristics shown in Table 2 below.

By analyzing the genotypes of the five genes as described above, we designed a method to simultaneously analyze five types of single nucleotide sequence variations using multiplex PCR using primers with a Miso-Winglet structure on one allele of each gene in order to predict the content of intramuscular fat and carcass weight for beef yield and meat quality in cattle by genotype.

In more detail, the FASN16024 gene used a Miso-Winglet structural primer (SEQ ID NO: 7) for allele A, and a 50-base oligonucleotide was added to the transposition region to distinguish the size of the amplified product from allele G; the SCD10329 gene used a Miso-Winglet structural primer (SEQ ID NO: 10) for allele C, and a 50-base oligonucleotide transposition region to distinguish the size of the amplified product from allele T; the FASN17924 gene used a Miso-Winglet structural primer (SEQ ID NO: 13) for allele A, and a 40-base oligonucleotide was added to the transposition region to distinguish the size of the amplified product from allele G; The ACACA2274 gene used a Miso-Winglet structure primer (SEQ ID NO: 16) for allele A, and a 40-base oligonucleotide was added to the transposition region to distinguish the size of the amplified product from allele G; the PLAG1 gene used a Miso-Winglet structure primer (SEQ ID NO: 19) for allele G, and a 30-base oligonucleotide was added to the transposition region to distinguish the size of the amplified product from allele C.

The above primer sequences are represented by SEQ ID NOs: 7 to 21. Primers for the entire base sequence of SEQ ID NOs: 7 to 21 were synthesized (Cosmogene Tech Co., Ltd.). Information on each primer and the size of the amplified product, etc. are presented in detail in Table 3 below.

(4) Setting Multiplex PCR conditions

The PCR reaction solution was prepared by mixing 1 ㎕ (20 ng/㎕) of the genomic DNA extracted from each beef in the above Example (a), 10 ㎕ of Simple Step 2X Multiplex PCR Master Mix, and 3 ㎕ of the mixed primer mixture, and then adding sterilized distilled water to make a final volume of 20 ㎕. The optimized PCR reaction temperature and time were determined to be 95 °C for 15 minutes once, 35 cycles of 95 °C for 20 seconds, 60 °C for 30 seconds, and 72 °C for 60 seconds, and then 72 °C for 3 minutes once. To confirm the final reaction product, 5 ㎕ of the PCR reaction product was loaded onto a 3% agarose gel, electrophoresis was performed at 250 V for 30 minutes, and the results were confirmed with a UV trans-illuminator.

(5) Results: Step of analyzing single nucleotide polymorphism; Beef genotype analysis

Multiplex PCR was performed on three types of Korean cattle and five types of imported cattle under the conditions of the above example (b-2), and the results are shown in Fig. 6.

As can be confirmed in Fig. 6, in Hanwoo 1, only 430 bp of the G allele was amplified for the FASN 16024 gene, confirming it as a G genotype homozygote, in the SCD 10329 gene, both 380 bp of the C allele and 330 bp of the T allele were amplified, confirming it as a C/T genotype heterozygote, in the FASN 17924 gene, only 240 bp of the G allele was amplified, confirming it as a G genotype homozygote, in the ACACA 2274 gene, only 160 bp of the G allele was amplified, confirming it as a G genotype homozygote, and in the PLAG1 gene, only 100 bp of the C allele was amplified, confirming it as a C genotype homozygote. In addition, Hanwoo 2 confirmed a genotype with the same result as Hanwoo 1.

In Hanwoo 3, the FASN 16024 gene was confirmed as a G genotype homozygote in which only 430 bp of the G allele was amplified, in the SCD 10329 gene, both 380 bp of the C allele and 330 bp of the T allele were amplified, confirming a C/T genotype heterozygote, in the FASN 17924 gene, only 240 bp of the G allele was amplified, in the ACACA 2274 gene, both 200 bp of the A allele and 160 bp of the G allele were amplified, confirming an A/G genotype heterozygote, and in the PLAG1 gene, both 130 bp of the G allele and 100 bp of the C allele were amplified, confirming a G/C heterozygote.

In Imported Cow 1, the FASN 16024 gene was confirmed as a G genotype homozygote in which only 430 bp of the G allele was amplified, the SCD 10329 gene was confirmed as a C genotype homozygote in which only 380 bp of the C allele was amplified, the FASN 17924 gene was confirmed as a G genotype homozygote in which only 240 bp of the G allele was amplified, the ACACA 2274 gene was confirmed as a G genotype homozygote in which only 160 bp of the G allele was amplified, and the PLAG1 gene was confirmed as a G/C heterozygote in which both 130 bp of the G allele and 100 bp of the C allele were amplified.

In imported cow 2, the FASN 16024 gene was confirmed as a G genotype homozygote in which only 430 bp of the G allele was amplified, the SCD 10329 gene was confirmed as a C genotype homozygote in which only 380 bp of the C allele was amplified, the FASN 17924 gene was confirmed as an A/G heterozygote in which both 280 bp of the A allele and 240 bp of the G allele were amplified, the ACACA 2274 gene was confirmed as a G genotype homozygote in which only 160 bp of the G allele was amplified, and the PLAG1 gene was confirmed as a G genotype homozygote in which only 130 bp of the G allele was amplified.

In imported cow 3, the FASN 16024 gene was confirmed as a G genotype homozygote in which only 430 bp of the G allele was amplified, the SCD 10329 gene was confirmed as a C genotype homozygote in which only 380 bp of the C allele was amplified, the FASN 17924 gene was confirmed as an A/G heterozygote in which both 280 bp of the A allele and 240 bp of the G allele were amplified, the ACACA 2274 gene was confirmed as an A/G genotype heterozygote in which both 200 bp of the A allele and 160 bp of the G allele were amplified, and the PLAG1 gene was confirmed as a G genotype homozygote in which only 130 bp of the G allele was amplified.

In imported cow 4, both 480 bp of allele A and 430 bp of allele G were amplified in the FASN 16024 gene, confirming it as an A/G heterozygote. In the SCD 10329 gene, only 380 bp of allele C was amplified, confirming it as a C genotype homozygote. In the FASN 17924 gene, only 240 bp of allele G was amplified, confirming it as a G genotype homozygote. In the ACACA 2274 gene, only 160 bp of allele G was amplified, confirming it as a G genotype homozygote, and in the PLAG1 gene, only 130 bp of allele G was amplified, confirming it as a G genotype homozygote.

In imported cow 5, both 480 bp of allele A and 430 bp of allele G were amplified in the FASN 16024 gene, confirming it as an A/G heterozygote. In the SCD 10329 gene, only 330 bp of allele T was amplified, confirming it as a T genotype homozygote. In the FASN 17924 gene, only 240 bp of allele G was amplified, confirming it as a G genotype homozygote. In the ACACA 2274 gene, only 160 bp of allele G was amplified, confirming it as a G genotype homozygote, and in the PLAG1 gene, only 130 bp of allele G was amplified, confirming it as a G genotype homozygote.

The genotypes of each sample (Korean cattle 1 to imported cattle 5) confirmed as above are summarized in Table 4 below.

From the above results, two types of genotypes were confirmed in three types of Korean cattle, and different genotypes were confirmed in all five types of imported cattle.

Through the examples so far, it has been confirmed that when the primer of the Miso-Winglet structure according to the present invention is used, it exhibits an excellent effect in that each allele of several multi-base sequence mutations can be analyzed with a single Multiplex PCR. In particular, since the size of a specific target product can be freely designed, it has a great advantage in that the genotype can be easily and quickly classified during analysis.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

Attach electronic file of sequence list

Claims (10)

(A) Primer-1 for allele-1, which includes a terminal base X of the mutation site of an allele and has a 5'-direction length of 10 to 30 bases, and a 5'-direction length of the latter region having a sufficient size to be distinguished from a PCR product by primer-2, wherein 1 to 3 consecutive bases in the 5'-direction of the latter region have a mutation part A that is non-complementary to the template, and the remaining bases are complementary to the bases of allele-1; and (B) Primer -2 for allele-2, which includes a terminal base X' of the mutation site of an allele and has a 5'-direction length of 10 to 30 bases and is complementary to the template.
In the first paragraph,
A primer pair for allele analysis, characterized in that 1 to 3 consecutive bases in the rear region of the primer-1 or 1 or 2 positions of the primer-2 are non-complementary to the template.
A method for analyzing alleles of single nucleotide sequence mutations by PCR using a primer pair according to claim 1 or 2.
In the third paragraph,
A method for analyzing alleles of multiple single nucleotide sequence variations using primer pairs for each allele of the multiple single nucleotide sequence variations.
A method for diagnosing the origin of a biological product using the method according to Article 3.
In paragraph 5,
A method for diagnosing the origin of a biological product, wherein the biological product is a food, and the origin is characterized by at least one of a variety, a place of origin, a producer, and a producing individual.
In paragraph 5,
A method for diagnosing the origin of a biological product, wherein the biological product is a pathogenic organism and the origin is a genotype of the pathogenic organism.
A method for predicting the characteristics of crops or livestock using the method according to Article 3.
A PCR reaction composition comprising a primer pair according to claim 1 or 2.
A kit for allelic analysis of single nucleotide sequence mutations comprising a primer pair according to claim 1 or 2.