KR100936133B1 - Method for quantifying and analyzing copy number variation in porcine KIT by an Oligonucleotide Ligation Assay - Google Patents

Abstract

The present invention relates to a method for quantitative analysis of KIT gene copy number variation using an oligonucleotide linkage assay. More specifically, the present invention is a KIT using oligonucleotide linkage assay Splicing mutation in the replication variability analysis method, copy number variation of the KIT gene duplicated in gene duplication is said oligonucleotide as determined by nucleotide connection method, KIT gene KIT characterized in that the measurement by the sequencing method of a conventional pie A method for quantifying gene copy number variation, and genotyping method for pig groping-related KIT genes, characterized in that the genotype is classified according to the KIT gene copy number variation measured by the method.

Since the method of the present invention can quantify KIT gene copy number variation more accurately and precisely than the conventional method, the method of the present invention is very useful for detecting KIT gene duplication status and genotyping.

KIT gene, copy number variation, genotyping

Description

Method for quantifying and analyzing copy number variation in porcine KIT by an Oligonucleotide Ligation Assay}

The present invention relates to a method for quantitative analysis of KIT gene copy number variation using an oligonucleotide linkage assay.

Differentiation of pigs is usually based on phenotypes such as groping and body shape, which are external features. However, even if the pig breed is distinguished by phenotype, it is almost impossible to identify the breed of the pig when it is converted to meat form after slaughter. In addition, in the current breeding system that produces F2, such as three-way hybrids for quality improvement, the preservation of unique characteristics such as the reliability of the sows used for hybridization has been a problem. In order to eliminate these problems, it is necessary to select and utilize DNA markers that can clearly distinguish pig breeds. Currently, in order to develop breed-specific DNA markers that appear in pigs, it is actively targeting various genes present in genomic DNA and mitochondrial DNA (mtDNA).

Among them, KIT (mast / stem cell growth factor receptor) among the color-related genes located on genomic DNA is a gene that regulates dominant white and color of pigs and is used as a very important DNA marker in distinguishing breeds of pigs. The Dominant white / KIT locus has been reported to be located on SSC8 (Johansson-Moller et al., Mamm Genome , 7: 822-8, 1996; Hirooka et al., J Hered , 93: 1, 2002). The white phenotype of pigs is determined by two KIT mutations: gene duplication is associated with an incomplete dominant phenotype, and splicing mutations induce a complete dominant allele (Johansson-Moller et al. , Mamm Genome. , 7: 822-8, 1996; Marklund, S. et al., Genome Res ., 8: 826-833, 1998). In the KIT locus, the main alleles are the recessive i allele of the color phenotype, the I ^P allele of the patch phenotype, the dominant I allele of the white phenotype, and the I ^Be allele of the belt phenotype. (allele) (Giuffra, E. et al., Mamm Genome. , 10: 1132-113, 1999; Johansson M et al., Genomics. , 14: 965-96, 1992). Pielberg et al. ( Genome Res . 13: 2171-217, 2003) classified I allele diversity and I allele as I ¹ , I ² , I ³ and I ^L allele. Dominant white gene I is as completely dominant with respect to the alleles I ^P and i Rend race (Landrace) and la charge white (Large White) may possess one or more of the I (I / I, I / I ^P, or I / i .) white hair (Marklund, S. et al., Genome Res ., 8: 826-833, 1998). Duroc, the most frequently bred breed of domestic hair, is i / i . In addition, Berkshire and Hampshire are estimated to be i / i (Marklund, S. et al., Genome Res ., 8: 826-833, 1998).

Current analysis methods of KIT loci include RFLP (Marklund, S. et al., Genome Res ., 8: 826-833, 1998), mini-sequencing and real-time PCR (Pielberg). , G. et al., Genetics ., 160: 305-3, 2002) and invaders and pyrosequencing assays (Pielberg, G. et al., Genome Res . 13: 2171-217, 2003). Among these, the pyro sequencing method is more accurate than real-time PCR or invader technology, and is considered to be the most powerful method for quantifying KIT duplication. However, the pyro sequencing method has a disadvantage in that the result is changed even by a single copy, and thus it becomes difficult to accurately classify the genotype as the number of copies increases. This is associated with an increase in the signs that the overlapping point becomes smaller (Pielberg, G. et al., Genome Res . , 13: 2171-217, 2003). Therefore, the detection of the approximate overlap ratio using the pyro sequencing analysis has a disadvantage in that it is difficult to accurately determine the genotype for samples that do not know the phylogenetic information including the parental genotype.

Therefore, the present inventors are studying a method to more accurately measure the copy number variation of the pig hunt-related KIT gene, and can detect a high copy number KIT duplication using quantitative oligonucleotide Ligation Assay. The present invention was completed by developing a method and confirming that the method is very accurate and accurate compared to the conventional pyrosequencing assay.

Accordingly, it is an object of the present invention to provide a copy variation analysis method for duplication of pig-related KIT genes using oligonucleotide linkage assays.

In addition, another object of the present invention is to measure the copy number variation of the KIT gene duplication using an oligonucleotide linkage assay, characterized in that the copy number variation of the splicing mutation of the KIT gene is measured using a pyro sequencing assay To provide a quantitative method for mutating the replication of the KIT gene.

It is also an object of the present invention to provide a genotyping method for a pig groping-related KIT gene, characterized in that the genotype is separated by the copying variation of the KIT gene quantified by the above method.

It is also an object of the present invention to provide a primer sequence useful for the analysis of copying variation of pig groping-related KIT gene duplication using the oligonucleotide linkage assay.

In order to achieve the object of the present invention as described above, the present invention provides a copy variation analysis method of the pig groping-related KIT gene duplication using an oligonucleotide linkage assay.

The invention also KIT gene, characterized in that for measuring the oligonucleotide is copy number variation of the KIT gene duplicated using a nucleotide connection method, and the measurement by scanning copy number variation of splice mutations in the KIT gene using the sequencing method Pyro It provides a method for quantifying replication variation in.

In addition, the present invention provides a method for genotyping of a pig groping-related KIT gene, characterized in that the genotype is separated by the copying variation of the KIT gene quantified by the method.

In addition, the present invention provides a primer sequence useful in a method for analyzing the copy variation of pig groping-related KIT gene duplication using the oligonucleotide linkage assay.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for analyzing duplication of KIT genes associated with swine groping using quantitative oligonucleotide ligation assay (qOLA).

Oligonucleotide linkage analysis is a method used to bind two adjacent oligonucleotide probes (Probe) by using DNA ligase (DNA Ligase).

To this end, in the present invention, a mutation in the L1MC1 / L1ME1 region in which a duplication breakpoint of the KIT gene, which is closely related to the currently reported pig search, is used.

Preferably, the replication variation analysis method of the KIT gene duplication of the present invention (a) PCR amplification of the DNA sample to be analyzed using a primer specific to the L1MC1 / L1ME1 region;

(b) treating the PCR amplification product of step (a) with a protease; And

(c) Amplification of the product treated with the protease in step (b) with a primer conjugated with fluorescent material, and copy number variation of the gene duplication (C / G) occurring at the overlapping point associated with the KIT locus (copy number) measuring the variation).

The DNA sample of step (a) can be separated from the biological sample. Such biological samples include solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, hair roots, tissue cultures or cells derived therefrom and their progeny. The samples also include reagent treatment, solubilized samples or cultured cells, cell supernatants and cell lysates. Preferably, the DNA sample may be isolated from pig blood or hair roots.

Isolation of DNA from the biological sample described above can be performed according to methods known in the art. For example, the isolation of genomic DNA from blood was performed in the following manner. Inverting several times after adding 10ml of blood (0.5M EDTA) and 40ml of buffer A (0.32M Sucrose, 1mM Tris-HCl pH7.5, 5mM MgCl ₂ , 1% Triton X-100 in 1L) Red blood cells were destroyed, and white blood cells were precipitated by spin down at 4 ° C. at 3,000 rpm for 10 minutes. After removing the supernatant, 15 ml of buffer A was added again, and then inverted. After spin down at 3,000 rpm for 10 minutes at 4 ° C., white blood cells were precipitated, and the supernatant was removed. After adding 4.5ml of pH8.0, 10mM Tris-HCl pH8.0 and 500ul of buffer C (5% SDS, 2mg / ml proteinase K), overnight treatment in 50 ℃ water bath It was. After strongly inverting by adding 1.35 ml of 6M NaCl to the culture solution, spin down for 15 minutes at 3000 rpm to carefully transfer the supernatant to a new tube, and then treated with 2 volumes of 100% ethanol. DNA was harvested using a pipette-tip-hook. After drying until the alcohol evaporated at room temperature, it was used by dissolving in 1 ml of TE buffer (10 mM Tris HCl, pH8.0; 2.5 mM EDTA).

Genomic DNA extraction from hair roots was performed in the following manner. Cut hair roots around 5 ~ 10 strands with good hair root attachment into proper length, add 100ul of 5% chelex and 1ul of 1mg / ml proteinase K, and then overnight in 55 ℃ water bath. overnight). The sample was boiled in boiling water for 15 minutes and then cooled completely at room temperature, followed by spin down at 3000 rpm for 5 minutes to use the supernatant for PCR reaction.

Primers capable of specifically amplifying the L1MC1 / L1ME1 region in step (a) may include 18 to 25 bp of each polynucleotide consecutively appearing only in the L1MC1 / L1ME1 region.

Preferably, primers capable of specifically amplifying the L1MC1 / L1ME1 region include a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 or a primer having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 3 It can be a set.

The very small amounts of SEQ ID NOs 2 and 3 form the basic product, and the actual amplification proceeds to SEQ ID NO 1 and TailR primers (SEQ ID NO: 11). In other words, SEQ ID NO: 2 and SEQ ID NO: 3 having different bases are attached to a common virtual sequence (including TailR) at the 5 ′ end of a primer made of a genomic sequence. After amplifying the degree products, SEQ ID No. 1 and TailR, a fictitious primer, are reacted so that each product amplified in small amounts is exponentially amplified.

TailR primer: 5`-CGT TCG TAC GAG AAT CGC T-3` (SEQ ID NO: 11)

PCR amplification in step (a) is preferably performed 27 times (for hair root-derived DNA) or 25 times (for blood-derived DNA) for 20 seconds at 94 ° C, 20 seconds at 58 ° C, and 30 seconds at 72 ° C. Can be done.

Proteolytic enzyme in step (b) may be processed for the purpose of removing the activity of Taq DNA polymerase (Polymerase). Examples of the protease include, but are not limited to, proteinase K, proteinase A, proteinase B, proteinase C, and the like. have.

Primer conjugated with the fluorescent material in step (c) can be designed based on the overlapping point of the KIT gene. In one embodiment of the present invention, the common primer (BPT_Com) has a base sequence represented by SEQ ID NO: 4 including a base from -24 ^th nucleotide to -1 ^st position from the overlapping point, and duplicates (BPT1) and normal replication ( Each reverse primer (SEQ ID NO: 5 and SEQ ID NO: 6) for BPT2) was designed and used respectively.

In addition, the 5 'end of the BPT_Com in the primer was labeled with fluorescein (fluorescein), phosphate group (phosphate group) was added to the 5' end of BPT1 and BPT2. In addition, (dA) ₁₀ and (dA) ₁₅ were added to the 3 'ends of BPT1 and BPT2 to distinguish the two qOLA products and to separate them from the peaks of the primers not used in the size differentiation step.

Amplification in the step (c) is preferably performed by repeating 30 seconds at 94 ℃ and 1 minute 30 seconds at 50 ℃ 10 times.

In the step (c), the copy number variation of the gene duplication (C / G) occurring at the overlapping point associated with the KIT locus is 3 ′ of the BPT_Com primer labeled with fluorescein using a ligase. BPT1 and BPT2 primers with a phosphate group added to their ends are used for phosphodiester bonds, and a sequence length of the overlapped gene and a normal gene using a sequencer (ABI3100) is used. It can be determined by dividing the difference of (base length) by dividing the value of the overlapping gene by the total (duplicate + normal) value.

Compared with the conventional pyro sequencing method to verify the precision and accuracy of qOLA_CNV according to the present invention, the standard curves showed good linearity in both qOLA_CNV and Pyro_CNV, but the RMS (5.05) and SD ( 1.04) was found to be more than twice as high as 2.09 and 0.45 of qOLA_CNV (see Example 5). Thus, it was found that the method of the present invention is more precise than the conventional pyrosequencing analysis.

In particular, the qOLA_CNV of the present invention has a more accurate analysis ability to detect KIT duplication of high replication number (> 4 per diploid genome).

On the other hand, we used a point mutation that occurs in the selective splicing region for high reliability for individuals without lineage information. That is, the above-described qOLA assay was used to detect the copy number variation of KIT gene duplication in the L1MC1 / L1ME1 region including the overlapping point, and the splice in the selective splicing region of the KIT gene. The previously reported pyro sequencing analysis method (Pielberg, G. et al., Genetics ., 160: 305-3, 2002) was used to detect the copy number variation.

Accordingly, the present invention comprises the steps of: (a) PCR amplifying a nucleic acid sample to be analyzed using a primer specific for the splicing mutation region of the KIT gene;

(b) measuring a copy number variation of the splicing mutation of the KIT locus by performing a pyro sequencing analysis on the PCR product of step (a); And

(c) copy number variation of gene duplication (C / G) occurring at the overlapping point associated with the KIT locus measured according to the method of claim 1 and copy number variation of the splicing mutation measured in step (b) It provides a method for quantifying the copy number variation of the pig groping-related KIT gene comprising the step of comparing and comparing each other.

The splicing mutation region of the KIT gene in step (a) refers to between exon 17 and intron 17 of the gene (Marklund, S. et al., Genome Res ., 8: 826-833, 1998). Primers capable of specifically amplifying the region may be, for example, but not limited thereto, having a nucleotide sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10.

The splicing mutation in step (b) preferably means that the first base of intron 17 of the KIT gene is replaced with adenine (A) in guanine (G).

The copy number variation of the splicing mutation in step (b) can be measured by measuring the area ratio of adenine in the total area of adenine (A) and guanine (G) after pyro sequencing using a probe primer.

In the step (c), pig groping is performed by combining the copy number variation of the KIT gene duplication measured by the qOLA assay of the present invention with the selective splice form variation of the KIT gene measured by the pyro sequencing. Quantify the copy number variation of the relevant KIT gene.

Furthermore, the present invention provides a method for genotyping of a pig hunt-related KIT gene, characterized in that the genotype is separated by copy number variation of the KIT gene quantified by the above method.

The genotyping may be performed by statistical analysis using a classification of coordinate analysis based on grouping of observed features and a method of nearest centroid sorting.

In one embodiment of the present invention, genotyping of 145 two F ₁ with lineage information was performed by combining qOLA_CNV and Pyro_Splice method for quantitative analysis of splicing forms of KIT replication (Example 7) Reference).

As a result, according to the method of the present invention, KIT It was confirmed that genotypes can be accurately classified (see Table 2 and FIG. 5). These results show that the genotyping method based on the combination of qOLA_CNV and Pyro_Splice can be applied to random populations without high reliability and systematic information.

Therefore, in another embodiment of the present invention, the present inventors performed genotyping of 100 commercial pigs and 159 large white pigs randomly selected by combining qOLA_CNV and Pyro_Splice methods (see Example 8). . As a result, the method of the present invention showed a diagnostic result that was completely consistent with the theoretical genotype. In addition, the method of the present invention was sensitive to the DNA derived from the hair roots, it can be seen that it can be applied to any DNA (see Fig. 6 and Table 3).

Furthermore, the inventors performed qOLA_CNV or Pyro_CNV on 100 randomly selected commercial pigs (CP) and 159 lazi white pigs (LW) and compared the two methods (see Example 9). As a result, the coefficient of variation of Pyro_CNV was more than twice higher than qOLA_CNV (see Table 4). The genotyping of 159 LW genotypes showed that qOLA_CNV showed clear results in genotyping, whereas the data of Pyro_CNV had many variations (CV range: 7.3-15.3%). Therefore, qOLA_CNV is better and more accurate than Pyro_CNV.

As described above, since the method of the present invention can quantify KIT gene copy number variation more accurately and precisely than the conventional method, the method of the present invention is very useful for detecting KIT gene duplication status and genotyping. .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1>

Extraction of Laboratory Animals and DNA Samples

The KIT alleles of 145 two F ₁ , which crossed Korean native pigs and Rendezvous crosses, were isolated and used to prove the reliability of qOLA_CNV. In addition, we randomly selected 100 commercial pigs (CP) with ternary crosses and 159 LWs using Duroc, Landrace and Large White (LW) species. KIT CNV experiments were performed and genotype analysis was performed for the KIT locus.

Genomic DNA was extracted from the blood of F ₁ and CP by the red cell lysis-proteinase K method (red cell lysis-proteinase K, Miller, SA. Et al., Nucleic Acids Res, 16:12, 1988). For LW, LW hair root DNA was prepared using 5% Chelex (Walsh, PS. Et al., Biotechniques , 10: 506-5, 1991). DNA recovered from the blood was dissolved in TE buffer (pH 8.0) and the hair root DNA was used as a PCR template strand.

<Example 2>

KIT  Confirmation of specificity of primers used for analysis of duplication

In the present invention, previously reported PCR primers were used for analysis of KIT CNV (Pielberg, G. et al., Genome Res . , 13: 2171-217, 2003). The primer sequence was selected at the KIT overlap point located at the repeating L1MC1 and L1ME1 (see a in FIG. 2). Forward primer (KITBPF) (SEQ ID NO: 1) showed 80% sequence identity with the L1MC1 sequence, and KIT1BPR (SEQ ID NO: 2) for normal CNV and KIT2BPR (SEQ ID NO: 3) reverse primer for overlapping CN1 with L1MC1 L1ME1 showed 63.2% and 94.7% sequence identity. This feature, in turn, raises the question that PCR amplification products may contain partially nonspecific ones in other genomic regions. To solve the question of the specificity of PCR primers, 27 hybrid clones were analyzed by somatic cell hybrid panel mapping in a panel formed from vectors before quantitative analysis (Yerle, M. et al. , Cytogenet Cell Genet , 73: 194-2, 1996).

PCR reaction of panel not cross clone 25ng template strand to full volume 25㎕ DNA, 1 ㅧ reaction buffer, each of forward and reverse primers of 20pmol (KITBPF and KIT1BPR; KITBPF and KIT2BPR), 100 u M dNTPs, and Taq DNA polymerase 5unit (GenetBio, Korea) was added. PCR results were analyzed using the INRA (http://www.toulouse.inra.fr/lgc/pig/pcr/pcr.htm) web page.

As a result of the experiment, 01000 11101 11000 11000 01000 10 of normal replication and 00000 11101 11010 11000 01000 of duplicated replication 10. KIT locus indicating the nonspecific amplification of the primer set used (assignment probability / correlation: SSC8p11 with 0.8789 / 0.9250 for normal and 0.8791 / 0.9250 for duplicate) was selected. As shown in Figure 3, the primer set was confirmed to be clear and accurate amplification.

<Example 3>

KIT  QOLA (qOLA_CNV) for resolving duplicates

<3-1> Design of primer for qOLA_CNV

QOLA_CNV primers were designed to perform qOLA to analyze KIT duplication (FIG. 2B). The category of consensus primers (BPT_Com: 5'-Fluorescein-GGC TAC ATA CTG TAT GAT TCC AA-3 ', SEQ ID NO: 4) ranges from -overlap to -1 ^st position in -24 ^th nucleotides. Each reverse primer for duplication and normal replication (BPT1: 5'-Phosohate-GGG TCA TGG CTT GAA AAA GAA AAA AAA AAA-3 ', SEQ ID NO: 5 and BPT2: 5'-Phosphate-CGA TAT GAC ATT CTG GAA ATA AAA AAA AAA AAA AA-3 ', SEQ ID NO: 6) was designed at the point of overlap.

The 5 'end of BPT_Com was labeled with fluorescein, and phosphate groups were added to the 5' ends of BPT1 and BPT2. In addition, (dA) ₁₀ and (dA) ₁₅ were added to the 3 'ends of BPT1 and BPT2 to distinguish the two qOLA products and to separate them from the peaks of the primers not used in the size differentiation step.

<3-2> qOLA-CNV

PCR was performed on each reverse transcription primer KIT1BPR (SEQ ID NO: 2) or KIT2BPR (SEQ ID NO: 3) containing 20 ng genomic DNA, 10 pmol KITBPF (SEQ ID NO: 1) and tail primer, 0.1 pmol tail primer binding site. ), 200 u M dNTPs, 1 μs reaction buffer and 2.5 unit Taq polymerase (GenetBio, Korea). Thermal conditions were repeated 27 times (for hair root-derived DNA) or 25 times (for blood-derived DNA) for 20 seconds at 94 ° C, 20 seconds at 58 ° C, and 30 seconds at 72 ° C.

To each PCR product obtained above, Proteinase K was added to a final concentration of 10 u g / μl and reacted at 55 ° C for 40 minutes.

qOLA is a PCR product treated with proteinase K, 1 μl, 1 μs reaction buffer, 1.5 unit Ampligase (EPICENTRE, USA), 1 pmol BPT_Com (SEQ ID NO: 4) and 0.5 pmol BPT1 primer (SEQ ID NO: 5) or BPT2 primer It was carried out using a 10 μl mixture containing (SEQ ID NO: 6). The thermal conditions of qOLA were repeated 10 times for 30 seconds at 94 ℃ and 1 minute 30 seconds at 50 ℃. 10 μl of deionize formamide was then added and analyzed by ABI Prism 3100 Genetic Analyzer, Applied Biosystems, USA. GeneScan software version 2.1 (Applied Biosystems, USA) was used to analyze the height and area corresponding to the peaks. Data collection and basic statistical calculations were performed using a Microsoft Excel program (Microsoft, USA).

Clones were prepared using the pGEM-T vector (Stratagen, USA) of each PCR product amplified by the two sets of primers SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 1 and SEQ ID NO: 3. The amplification products of the clones were amplified again using M13 forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8), and purified to dilute duplicate and normal replication sequentially from 0% to 100%. qOLA_CNV performed 4 repetitive PCRs and estimated two standard curves for the height and area of the peak (a and b of FIG. 4). In both the height and area, the correlation coefficient was 0.999 in the standard curve, indicating a very good linearity.

Correlation coefficients only show the linearity of the standard curve. To compare precision and accuracy, the root mean square (RMS) and standard deviation (SD) were compared. RMS, which represents the bias of the baseline ratio, and SD, which represents the amount of variation in baseline replication, are a good indicator of finding observation data that match the expectations better than the correlation coefficient. The expected peak height in TqOLA_CNV fits better with the reference value and showed the lowest variation (Table 1). In particular, quantitative analysis for accurate genotyping of individuals with four or more KIT copies in their entirety requires more accurate assays in the region between 60% and 90% of the standard curve. For qOLA_CNV, the peak areas were 2.21 and 1.17 for bias and RMS, respectively, in the region between 60% and 90%. On the other hand, the peak heights in qOLA_CNV were 0.86 and 0.51 in the same zone.

<Example 4>

KIT  Pyrosequencing (Pyro_CNV) to analyze redundancy

PCR and pyro sequencing for quantitative analysis of KIT CNV were performed according to previously known methods using PyroMark MD (PyroMark MD, Biotage, Sweden) (Pielberg, G. et al., Genome Res. , 13: 2171-217, 2003).

Example 5

Comparative Analysis of Standard Curves by qOLA_CNV and Pyro Sequencing

Performed in Example 3 qOLA_CNV and Pyro_CNV performed in Example 4 were compared. The serial dilutions used for qOLA_CNV were used to estimate the standard curve of Pyro_CNV. As a result, the standard curve of Pyro_CNV showed good linearity (correlation coefficient 0.995) (FIG. 4C). However, the RMS (5.05) and SD (1.04) of the overall Pyro_CNV bias were more than doubled in comparison with the peak heights (2.09 and 0.45) of qOLA_CNV (Table 1). Therefore, it can be seen that the CNV estimation of the pig KIT using the peak height of qOLA_CNV is lower than the Pyro_CNV system error and variation (Fig. 4d).

Comparison of deflection, standard deviation, and RMS of the standard curve Reference ^a (%) | Bias | of qOLA_CNV (Height) SD of qOLA_CNV (Height) | Bias | of qOLA_CNV (Area) SD of qOLA_CNV (Area) | Bias | of Pyro_CNV SD of Pyro_CNV 0 0 0 0 0 3.64 0.38 10 1.55 0.51 1.91 0.24 1.13 1.30 20 3.43 0.42 3.67 0.46 5.87 0.31 30 4.01 0.56 3.31 0.93 4.48 0.69 40 3.09 0.31 1.92 1.08 7.17 1.04 50 2.32 0.58 3.40 1.95 7.48 1.49 60 1.27 0.61 1.65 1.32 5.23 0.97 70 0.51 0.33 2.07 0.93 3.75 0.93 80 0.56 0.72 2.91 1.34 1.04 0.92 90 0.87 0.13 2.01 1.03 3.62 1.81 100 0 0 0 0 6.98 0.52 RMS1 ^b 2.09 0.45 2.16 1.03 5.05 1.04 RMS2 ^c 0.85 0.51 2.21 1.17 3.73 1.21

a: target value described as expected of replicated copy on X axis of FIG. 4

b: total RMS

c: RMS of area between 60-90%

<Example 6>

In the form of a splice (Pyro_Splice) KIT  Quantitative Analysis of Replication

KIT21 and KIT35 PCR primer sets (SEQ ID NO: 9 and SEQ ID NO: 10) and thermal conditions were performed according to previously known methods (Marklund, S. et al., G enome Res 1998, 8: 826-8). Thermal conditions were repeated 30 times 20 seconds at 94 ℃, 20 seconds at 63 ℃, and 30 seconds at 72 ℃.

Forward primer KIT21: SEQ ID NO: 9

5'-GTA TTC ACA GAG ACT TGG CGG C-3 '

Reverse primer KIT35: SEQ ID NO: 10

5'-AAA CCT GCA AGG AAA ATC CTT CAC GG-3 '

Pyro sequencing is described by Pielberg et al ( Genome Res . 13: 2171-217, 2003). Probe primers used for pyro sequencing Pielberg et al (Genetics 160 : 305-311, January 2002) as described in (5'-TAA TTA CIT GGT CAA AGG AAA C-3 '(I is Inosine, guanine) ), And prepared by vacuum preparation of Streptavidin beads (Streptavidin bead, Streptavidin Sepharose High Performance, Amersham), 2X binding buffer and PCR amplification products, followed by vacuum primer (Vacuum Prep) The mixture was dropped into a mixture of annealing buffer) and denatured at 80 ° C. for 2 minutes.

<Example 7>

Genotyping by combination of qOLA_CNV and Pyro_Splice methods

To demonstrate the isolation of KIT alleles, the Pyro_Splice method for the quantitative analysis of qOLA_CNV performed in Example 3 and KIT replication in the spliced form performed in Example 6 Was performed to genotype analysis of 145 two F ₁ cross-crossing Korean native pig and Renracedon (Landrace).

The combination of qOLA_CNV and Pyro_Splice methods could be used to analyze the genotypes of the parent generation and F ₁ populations. In the parent generation group, 19 Korean natives (8 males and 11 females) all had black phenotype homozygotes i / i , but 17 males (8 males and 9 females). Lendace possessed a combination of three different genotypes, the I ¹ , I ² and i alleles of the White phenotype (FIG. 5 a). The F ₁ population possessed three genotypes of heterozygotes I ¹ / i and I ² / i and homozygotes i / i , which are combinations of alleles inherited from the parent (FIG. 5 b).

Statistical analysis was performed using the classification of coordinate analysis based on grouping of observed features for genotyping and the nearest centroid sorting method of the PROC FASTCLUS procedure of SAS software. In other words, the statistical analysis sets the theoretical genotype splice and the overlapping rate to 12 kinds of seeds, assigning observations to the nearest class based on Euclidean distance, and classifying the class as the center class. Labeled as indicated.

As a result of genotyping, the coordinate analysis classification method and statistical analysis classification method were found to be 100% identical, and the same as the theoretical genotype ratio (Table 2). Alleles of F ₁ were correctly isolated from the lendace genotype of each parental generation and clustered at a determinable proportion consistent with the theoretical ratio (c and d in FIG. 5). These results show that the genotyping method based on the combination of qOLA_CNV and Pyro_Splice can be applied to random populations without high reliability and systematic information.

Theoretical Genotype of KIT Locus Using Splicing Mutations and Duplicate Features Genotypes ^a Numbers of splice / normal Ratios of splice (%) Copy numbers of duplication / normal (total copy numbers) Ratios of duplication (%) Seed No. ^b i / i ( I ^Be ) 0/2 0 0/2 (2) 0 One I ^P / i ( I ^Be ) 0/3 0 1/2 (3) 33.3 2 I ^P / I ^P 0/4 0 2/2 (4) 50 3 I ² / I ^P 1/4 20 3/2 (5) 60 4 I ^One / I ^P 1/3 25 2/2 (4) 50 5 I ² / i ( I ^Be ) 1/3 25 2/2 (4) 50 5 I ¹ / i ( I ^Be ) 1/2 33.3 1/2 (3) 33.3 6 I ² / I ² 2/4 33.3 4/2 (6) 66.7 7 I ^One / I ² 2/3 40 3/2 (5) 60 8 I ³ / I ^P 2/3 40 3/2 (5) 60 8 I ^One / I ^One 2/2 50 2/2 (4) 50 9 I ³ / i ( I ^Be ) 2/2 50 2/2 (4) 50 9 I ² / I ³ 3/3 50 4/2 (6) 66.7 10 I ^One / I ³ 3/2 60 3/2 (5) 60 11 I ³ / I ³ 4/2 66.7 4/2 (6) 66.7 12

a: The I ^L allele is not included and genotypes are sorted by the rate of splicing mutations.

b: Number of class centroids used for statistical genotyping.

<Example 8>

Analysis of Commercial and Large White Don by Combination of qOLA_CNV and Pyro_Splice Methods

A combination of qOLA_CNV and Pyro_Splice methods was used to perform genotyping of randomly selected 100 commercial pigs and 159 large white pigs. qOLA_CNV and Pyro_Splice were performed in the same manner as in Example 4 and Example 6.

First, in the case of 100 commercial pigs (CP), the terminating piglet Duroc consists of recessive homozygotes i / i , and F ₁ used as sows is phenotypicly white and homozygous or heterozygous. It is assumed to have a dominant I allele of. CP is therefore expected to have at least one i allele inherited from Duroc. The features observed by the combination of qOLA_CNV and Pyro_Splice methods were grouped into four categories: I ¹ / i , I ² / i , I ^P / i and i (I ^Be ) / i (FIG. 6B). The grouping of observed features and the statistical clustering were in perfect agreement.

For 159 large white pigs, the category of CNV (3-6 copies) in LW is expected to be wider than CP, which is an extended assessment of the qOLA_CNV analysis. Moreover, since LW DNA was prepared from the hair root, the sensitivity of qOLA_CNV can be estimated. First, coordinate analysis classification was performed; LW was classified into eight types including eleven genotypes based on grouping characteristics of data obtained by experiments (FIG. 6C).

Then, based on the classification, statistical analysis was performed using the nearest centroid sorting method of the PROC FASTCLUS procedure of SAS software. As a result, both genotyping methods showed the same results except for two individuals (IDs: T7183 and Y4410Q). In T7183 individuals, qOLA_CNV and Pyro_Splice were 63.4% and 62.9%, and the coordinates of the points were clearly distinguished from the I ¹ / I ³ standard coordinates (60% and 60%). However, the distance between the coordinates and the I ³ / I ³ centers (66.7% and 66.7%) was 4.78, slightly longer than 4.45 between the coordinates and the I ¹ / I ³ centers (Table 3).

Comparison of observations and statistics for 159 heads of Raji White Don 4 ^a 5 ^a 7 ^a 8 ^a 9 ^a 10 ^a 11 ^a I ² / I ^P 6 (8.77%) - - - - - - I ¹ / I ^P or I ² / i ( I ^Be ) - 12 (7.55) - - - - - I ² / I ² - - 28 (14.47) One (0.63) - - - I ¹ / I ² or I ³ / I ^P - - - 58 (36.48 - - - I ¹ / I ¹ or I ³ / i ( I ^Be ) - - - - 51 (32.08) - - I ² / I ³ - - - - - 5 (3.14) - I ^One / I ³ - - - - - - 2 (1.26) I ³ / I ³ - - - - - - One (0.63)

The genotypes designated by the observed grouping features are shown in rows and the number of class centroids for statistical analysis is shown in rows.

Two mismatched results between the two methods are shown in italics and bold.

a: number of seed numbers of each genotype

Example 9

Comparison of qOLA_CNV and Pyro_CNV in the Analysis of Commercial and Large White Money

QOLA_CNV and Pyro_CNV were performed on 100 commercial pigs (CP) and 159 ludge white pigs (LW), and the two methods were compared.

As a result, the coefficient of variation (CV) of 70 head I ¹ / i CP was 2.9% for qOLA_CNV and 6.9% for Pyro_CNV. The CV value of Pyro_CNV was more than twice higher than qOLA_CNV (Table 4). This is the same tendency as shown in the comparison of the standard curves by the two methods (Example 5).

In addition, 159 heads of LW were analyzed using genotypes of selected observations near the cluster seed using the FASTCLUS procedure, while qOLA_CNV showed clear results in genotyping, whereas Pyro_CNV data showed many variations (CV range). 7.3-15.3%). Therefore, qOLA_CNV is better and more accurate than Pyro_CNV.

Comparison of the Accuracy and Precision of qOLA_CNV, Pyro_CNV, and Pyro_Splice in Genotyping of Commercial and Lazi Whitedon Breed or Cross (Type of DNA) Estimated Genotype by qOLA No. of pigs qOLA (%) Pyrosequencing (%) Expected ^a Observed ^bd in qOLA_CNV CV ^c Expected ^a Observed ^bd in Pyro_CNV CV ^c Expected ^a Observed ^bd in Pyro_Splice CV ^c Commercial money (blood-derived DNA) I ^One / i 70 33.3 33.7 2.9 33.3 31.8 6.9 33.3 35.9 2.2 I ² / i 23 50 49.4 1.8 50 44.7 7.2 25 27.7 2.6 I ^P / i 5 33.3 34.3 na ^g 33.3 29.7 na 0 0 na i ( I ^Be ) / i 3 0 0 na 0 1.8 na 0 0 na Lazi Whitedon (hair rooted DNA) I ¹ / I ¹ or I ^{3 /} i ( I ^Be ) 51 50 50.7 3.4 50 47.1 11.7 50 51.4 2.1 I ¹ / I ² or I ³ / I ^P 58 60 58.2 3.4 60 54.6 15.9 40 41.8 2.4 I ¹ / I ^P or I ² / i ( I ^Be ) 12 50 51.0 3.1 50 47.4 7.3 25 27.9 3.8 I ² Of I ² 24 66.7 65.7 3.5 66.7 61.1 11.3 33.3 34.3 6.2 I ^One Of I ³ 2 60 57.3 na 60 58.3 na 60 61.2 na I ² Of I ^P 6 60 59.2 na 60 52.3 na 20 22.8 na I ² Of I ³ 5 66.7 64.5 na 66.7 60.3 na 50 50.4 na I ³ Of I ³ One 66.7 63.4 na 66.7 58.7 na 66.7 62.9 na

a: theoretical ratio corresponding to each genotype

b: mean value of the ratios observed for each genotype

c: coefficient of variation. Four genotypes, including individuals with more than 10 genotypes, were selected for analysis.

d: Oligonucleotide binding assay for KIT duplication

e: Pyro Sequencing Rate for KIT Redundancy

f: Pyro sequencing ratio for splice variation of KT

g: na means not analyzed

Figure 1 is a schematic of the comparison of the copy number variation detection method and pyro sequencing method of the pig groping-related KIT gene according to the present invention.

Figure 2 shows the overall schematic diagram (a) of the pig KIT locus and overlap and the base sequence (b) around the overlap point.

3 is a result of PCR analysis using a somatic cell hybrid panel.

a: Somatic hybrid panel results to verify the specificity of the SEQ ID NO: 1 and SEQ ID NO: 2 primer set.

b: Somatic hybrid panel results to verify the specificity of the SEQ ID NO: 1 and SEQ ID NO: 3 primer set.

The numbers in each lane indicate the number of PCR amplified clones among the clones (27 total) used in the somatic hybrid panel.

4 is a standard curve of qOLA_CNV and Pyro_CNV.

X axis of a, b, c: each expectation of duplicate copies

Y-axis of a and b: As a result of performing qOLA, the Y-axis of a represents the actual value of the peak height among qOLA_CNV observations for each expectation, and the Y-axis of b represents the actual peak area of the qOLA observations for each expectation. It is a value.

Y-axis of c: The actual values of Pyro_CNV are expressed as the values of the pyro sequencing.

d: RMS1 of Table 1, where X axis is | Bias | Y-axis represents the SD value. In other words, the X-axis represents the actual absolute deviation and the Y represents the variation in the iteration.

FIG. 5 shows genotyping results by separation analysis of KIT alleles using pyro sequencing (Pyro_Splice) for quantitative analysis of qOLA_CNV and splicing forms of KIT replication for KIT replication number analysis.

a and b: The qOLA result (qOLA_CNV) is written on the X-axis and the pyro sequencing result (Pyro_Splice) is written on the Y-axis. a is the ratio of parental pigs and b is the ratio of piglets, showing that they are grouped by genotype.

c and d: shows how the piglets are distributed in the genotypes of their respective piglets. The x-axis is the genotype of the parent and the y-axis is the ratio of the piglets. c is the qOLA distribution of the piglets and d is the pyro sequencing distribution. The parental pigs were Landrace on one side and Duroc (i / i) on the other, so the Duroc genotypes fixed on the X-axis of c and d were not shown.

e: shows how each genotype actually appears when performing qOLA and pyro sequencing.

Figure 6 shows the results of genotyping of commercial and large white pigs using a combination of qOLA_CNV and Pyro_Splice.

a: The 12 seed types are used for statistical analysis.

b and c: Coordinate analysis, showing that each result is concentrated around the seed. c's T7183 and Y4410Q are the two discrepancies in Table 3 (when comparing the Coordinate Analysis and Statistical Classification), b is a group of commercial pigs and c is a large white group.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Method for quantifying and analyzing copy number variation in          porcine KIT by an Oligonucleotide Ligation Assay <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer KITBPF <400> 1 caactatgct acatccaggc 20 <210> 2 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer KIT1BPR <400> 2 gtaaccgttc gtacgagaat cgctgtggct cttttgaggt cag 43 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer KIT2BPR <400> 3 gtaaccgttc gtacgagaat cgctctgtct ccatggtttt gcc 43 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer BPT_Com <400> 4 ggctacatac tgtatgattc caa 23 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer BPT1 <400> 5 gggtcatggc ttgaaaaaga aaaaaaaaaa 30 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer BPT2 <400> 6 cgatatgaca ttctggaaat aaaaaaaaaa aaaaa 35 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M13 forward primer <400> 7 cacgacgttg taaaacgac 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M13 Reverse primer <400> 8 ggataacaat ttcacacagg 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer KIT21 <400> 9 gtattcacag agacttggcg gc 22 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer KIT35 <400> 10 aaacctgcaa ggaaaatcct tcacgg 26 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TailR primer <400> 11 cgttcgtacg agaatcgct 19  

Claims (16)

(a) L1MC1 / L1ME1 region of the DNA sample to be analyzed using a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 or a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 3 PCR amplifying; (b) treating the PCR amplification product of step (a) with a protease; (c) the product treated with the proteolytic enzyme in step (b) is a primer set having a nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 4 and SEQ ID NO: 6 Amplifying with a primer set having a nucleotide sequence; And (d) measuring the copy number variation of the gene duplication (C / G) occurring at the overlapping breakpoint associated with the KIT locus of the PCR amplification product of step (c). Quantitative analysis of copy number variation of gene duplications. The method of claim 1, wherein the DNA sample of step (a) is isolated from pig blood or hair roots. delete The method according to claim 1, wherein the PCR amplification in step (a) is repeated 20 or 25 times at 94 ° C, 20 seconds at 58 ° C, and 30 seconds at 72 ° C. According to claim 1, wherein the proteolytic enzyme of step (b) is proteinase K (proteinase K), proteinase A (proteinase A), proteinase B (proteinase B), or proteinase C (proteinase C) is characterized in that How to. delete The method of claim 1, wherein in the step (c), the amplification is repeated 10 times 30 seconds at 94 ℃ and 1 minute 30 seconds at 50 ℃. delete delete delete delete The genotyping method of a pig groping-related KIT gene, characterized by separating alleles by combining the method of claim 1 and the pyro sequencing analysis method for quantifying the copy number variation of the splicing mutant of the KIT locus. 13. The method of claim 12, wherein the genotyping is performed using a classification of coordinate analysis based on the grouping of observed features and a statistical centroid sorting method. delete delete delete

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