KR102478811B1 - Novel biomarker composition for diagnosis of Alzheimer's Disease based on multiplex PCR platform - Google Patents

Abstract

The present invention relates to a novel marker composition for diagnosing Alzheimer's disease based on a multiplex PCR platform and its use, and specifically, the present invention relates to an Alzheimer's disease containing at least one gene selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7. A composition for a disease diagnostic marker, a composition for diagnosing Alzheimer's disease, a diagnostic kit, and a primer set for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for diagnosing Alzheimer's disease, and the present invention also relates to a composition for predicting or diagnosing the onset of Alzheimer's disease A method for providing information and a method for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for diagnosis of Alzheimer's disease. CDK11b, CDK2, CDK5rap3, and CDK7, which are diagnostic marker genes for Alzheimer's disease of the present invention, have increased expression levels specifically in the plasma of Alzheimer's-induced individuals compared to the normal group. It has the advantage of being able to easily diagnose the onset of, and when performing a multiplex polymerase chain reaction using the primer set for detecting biomarkers of the present invention, the above four types of biomarkers can be rapidly and accurately detected at the same time. There is an effect.

Description

Novel biomarker composition for diagnosis of Alzheimer's Disease based on multiplex PCR platform for diagnosis of Alzheimer's disease based on multiplex PCR platform}

The present invention relates to a novel marker composition for diagnosis of Alzheimer's disease based on a multiplex PCR platform and its use.

Degenerative brain disease refers to a disease that occurs in the brain among degenerative diseases that occur with age, and can be classified by considering the main symptoms and the part of the brain that is affected. Representatively, Alzheimer's disease or Parkinson's disease are included. .

Alzheimer's disease, a representative degenerative brain disease, is one of the diseases that cause dementia, and is a disease in which cognitive function including memory is progressively deteriorated due to a gradual onset as aging progresses. Although the exact pathogenesis of Alzheimer's disease is not known, it is currently known that the general mechanism is that beta-amyloid protein is made excessively and is deposited in the brain and has a harmful effect on brain cells.

Acetylcholinesterase (AChE) inhibitors or N-Methyl-D-aspartate (NMDA) receptor antagonists are used as drugs for the treatment of Alzheimer's disease, but most of these drugs are used for early-stage Alzheimer's disease patients. It is known to be effective against

In addition, methods such as a clinical exam and brain imaging are used to diagnose Alzheimer's disease, but it is very difficult to diagnose Alzheimer's disease in an early stage using such a diagnosis method.

In particular, through analysis of the brains of people with Alzheimer's disease, pathology such as the presence of extracellular aggregates of Amyloid-β peptide and abnormal nerve fiber entanglement of Tau, a microtubule protein However, there is a very large limitation in effectively preventing Alzheimer's disease because there is no clear biomarker that can confirm the possibility of Alzheimer's disease other than a tissue biopsy in which brain tissue must be directly collected.

Therefore, although early diagnosis of the disease is essential for effective treatment of Alzheimer's disease, there are currently few methods for accurately and quickly diagnosing Alzheimer's disease.

On the other hand, the cell cycle is a complex process involved through the regulation of various proteins so that cells can grow and proliferate. Usually, cells do not end after growing and dividing once, but the cells grow and divide again. have a regular cycle.

Among cell cycle processes, Cyclin-dependent kinases (CDKs) are serine-threonine kinases that are best known for regulating cell cycle progression. This cell cycle regulation is achieved by the activity through the combination of specific members of the CDK family and the cyclin family, and each CDK-cyclin complex regulates different cell cycle phases. In addition, certain CDK family members perform unique roles by phosphorylating various specific factors, including various steps such as transcription outside the cell cycle, neuronal migration, and synaptic plasticity. It is known to regulate biological processes of neuronal development. In addition, abnormal activity and dysregulation of certain CDK families are associated with viral infections and proliferative renal disease. And it is known to be found in many diseases of cancer (cancer), but there is no study yet that cyclin-dependent kinases (CDKs) can be used as biomarkers for Alzheimer's diagnosis.

1. US registered patent US8377640 2. European registered patent EP01724588

Accordingly, the present inventors, while conducting research to accurately and quickly discover new biomarkers for the diagnosis of Alzheimer's disease, measured the expression levels of four genes, CDK11b, CDK2, CDK5rap3, and CDK7, from blood-derived plasma samples. The present invention was completed by confirming that diseases can be effectively diagnosed.

Accordingly, an object of the present invention is to provide a composition for diagnosis of Alzheimer's disease containing at least one gene selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7.

Another object of the present invention is to provide a composition for diagnosing Alzheimer's disease comprising a substance for measuring the expression level of one or more genes selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7.

Another object of the present invention is to provide a primer set for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for diagnosis of Alzheimer's disease.

Another object of the present invention is to provide a kit for diagnosing Alzheimer's disease, including the diagnostic composition of the present invention.

Another object of the present invention is to provide an information providing method for predicting or diagnosing the onset of Alzheimer's disease.

Another object of the present invention is to provide a method for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for the diagnosis of Alzheimer's disease.

In order to achieve the above object, the present invention provides a composition for diagnosis of Alzheimer's disease containing at least one gene selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7.

In one embodiment of the present invention, the CDK11b consists of the nucleotide sequence of SEQ ID NO: 1, the CDK2 consists of the nucleotide sequence of SEQ ID NO: 2, the CDK5rap3 consists of the nucleotide sequence of SEQ ID NO: 3, and the CDK7 may consist of the nucleotide sequence of SEQ ID NO: 4.

In addition, the present invention provides a composition for diagnosing Alzheimer's disease comprising a substance for measuring the expression level of one or more genes selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7.

In one embodiment of the present invention, the material may be a primer or probe capable of specifically binding to CDK11b, CDK2, CDK5rap3 or CDK7 genes.

In one embodiment of the present invention, the primers capable of specifically binding to CDK11b are primer sets consisting of SEQ ID NOs: 5 and 6, and the primers capable of specifically binding to CDK2 are primers consisting of SEQ ID NOs: 7 and 8. A set of primers capable of specifically binding to CDK5rap3 may be a primer set consisting of SEQ ID NOs: 9 and 10, and primers capable of specifically binding to CDK7 may be a primer set consisting of SEQ ID NOs: 11 and 12.

In addition, the present invention provides primer sets for simultaneous detection of CDK11b, CDK2, CDK5rap3, and CDK7 for diagnosis of Alzheimer's disease, consisting of SEQ ID NOs: 5 to 12.

In addition, the present invention provides a kit for diagnosing Alzheimer's disease, including the composition for diagnosis of the present invention.

In addition, the present invention, (a) measuring the expression level of CDK11b, CDK2, CDK5rap3 or CDK7 gene from a biological sample of a patient suspected of having Alzheimer's disease; and (b) comparing the expression level of the gene with the expression level of the corresponding gene in a normal control sample.

In one embodiment of the present invention, as a result of measuring the expression level of the gene, when the expression level of the gene is increased compared to a normal control group, a step of determining that Alzheimer's disease is onset may be further added.

In one embodiment of the present invention, the biological sample may be blood, plasma or brain tissue.

In addition, the present invention, (a) extracting genomic DNA from a biological sample of a patient suspected of having Alzheimer's disease; And (b) performing a multiplex polymerase chain reaction using the extracted genomic DNA as a template and using all of the primer sets consisting of SEQ ID NOs: 5 to 12; CDK11b, CDK2 for diagnosing Alzheimer's disease, A method for simultaneous detection of CDK5rap3 and CDK7 is provided.

CDK11b, CDK2, CDK5rap3, and CDK7, which are biomarker genes for Alzheimer's disease according to the present invention, are specifically increased in expression in the plasma of subjects induced by Alzheimer's compared to the normal group, targeting plasma samples that are easy to obtain There is an effect of easily diagnosing the onset of Alzheimer's disease, and when performing a multiplex polymerase chain reaction using the primer set for detecting the biomarker, the four types of biomarkers can be quickly and efficiently tested in one polymerase chain reaction. and can be accurately detected at the same time.

1 shows the results of analyzing the expression levels of differentially expressed genes in plasma derived from the blood of Alzheimer's disease-induced mice and normal mice by FPKM.
2 shows the results of singular RT-PCR analysis using primer sets capable of detecting the biomarkers for Alzheimer's disease diagnosis (CDK11b, CDK2, CDK5rap3, and CDK7) discovered in the present invention.
Figure 3 shows the results confirming that CDK11b, CDK2, CDK5rap3 and CDK7 biomarkers can be simultaneously detected through a single multiplex-PCR (AdPlex PCR) using all the primer sets designed in the present invention.

The present invention provides novel uses of CDK11b (Cyclin Dependent Kinase 11B), CDK2 (Cyclin Dependent Kinase 2), CDK5rap3 (CDK5 Regulatory Subunit Associated Protein 3) and CDK7 (Cyclin Dependent Kinase 7) as novel biomarkers for the diagnosis of Alzheimer's disease. It is characterized by the fact that it was first identified.

Conventional methods for diagnosing Alzheimer's disease include neurocognitive testing, magnetic resonance imaging (MRI), and pathological analysis through brain tissue biopsy. There are problems that can put a lot of pressure on you.

Accordingly, the present inventors, while conducting research to discover new biomarkers that can diagnose Alzheimer's disease simply and quickly while relieving the inconvenience of tissue analysis, found CDK11b, CDK2, CDK5rap3 or It was found that the onset of Alzheimer's disease can be quickly and accurately diagnosed by measuring the expression level of the CDK7 gene.

In this regard, in one embodiment of the present invention, after separating blood from an Alzheimer's disease-induced mouse and a normal mouse, plasma was separated from the blood, total RNA was extracted from the plasma, and whole genome analysis was performed. Thus, genes showing differences in expression levels between the diseased and normal groups were screened, and the difference in differentially expressed expression levels was quantitatively analyzed.

As a result, it was confirmed that the levels of CDK11b, CDK2, CDK5rap3 or CDK7 RNA were significantly increased in the Alzheimer's disease group compared to the normal group.

Therefore, when Alzheimer's disease is induced, the expression of these genes is increased compared to normal, and this confirmation can be confirmed in tissues and blood, preferably in plasma.

Therefore, it was found that the presence or absence of Alzheimer's disease can be diagnosed simply and accurately by measuring the expression level of these genes in an easily obtainable plasma sample.

Therefore, the present invention can provide a composition for a diagnostic marker for Alzheimer's disease containing at least one gene selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7, preferably the CDK11b is composed of the nucleotide sequence of SEQ ID NO: 1 , The CDK2 may consist of the nucleotide sequence of SEQ ID NO: 2, the CDK5rap3 may consist of the nucleotide sequence of SEQ ID NO: 3, and the CDK7 may consist of the nucleotide sequence of SEQ ID NO: 4.

In addition, the present invention can provide a composition for diagnosing Alzheimer's disease comprising a substance for measuring the expression level of one or more genes selected from the group consisting of CDK11b, CDK2, CDK5rap3 and CDK7, wherein the substance is CDK11b, CDK2, CDK5rap3 or Primers or probes capable of specifically binding to the CDK7 gene may be used.

In one embodiment of the present invention, a primer set capable of detecting CDK11b, CDK2, CDK5rap3 or CDK7, which is a biomarker for diagnosis of Alzheimer's disease discovered in the present invention, was designed.

Specifically, primers capable of specifically binding to CDK11b are primer sets consisting of SEQ ID NOs: 5 and 6, primers capable of specifically binding to CDK2 are primer sets consisting of SEQ ID NOs: 7 and 8, and primers specific to CDK5rap3. Primers capable of specifically binding are primer sets consisting of SEQ ID NOs: 9 and 10, and primers capable of specifically binding to CDK7 are primer sets consisting of SEQ ID NOs: 11 and 12, and each of these primer sets are respectively It was confirmed that the gene could be accurately detected in a plasma sample of an individual with Alzheimer's disease.

When PCR was performed using the primer set consisting of SEQ ID NOs: 5 and 6 of the present invention, an amplification product of the CDK11b gene having a size of 398 bp of SEQ ID NO: 13 was confirmed, and the primers consisting of SEQ ID NOs: 7 and 8 of the present invention When PCR was performed using the set, an amplification product of the CDK2 gene having a size of 266 bp of SEQ ID NO: 14 was confirmed, and when PCR was performed using the primer set consisting of SEQ ID NOs: 9 and 10 of the present invention, the sequence The amplification product of the CDK5rap3 gene having a size of 114 bp of SEQ ID NO: 15 was confirmed, and when PCR was performed using the primer set consisting of SEQ ID NOs: 11 and 12 of the present invention, the CDK7 gene having a size of 178 bp of SEQ ID NO: 16 Amplification products were confirmed.

Therefore, the present invention can provide a primer set for simultaneous detection of CDK11b, CDK2, CDK5rap3, and CDK7 for diagnosis of Alzheimer's disease, consisting of SEQ ID NOs: 5 to 12.

Also, the expression level of the gene may preferably be the level of RNA expressed from the gene, and more preferably the level of mRNA.

In the present invention, the term “primer” refers to a single agent that can act as the starting point of template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature and in a suitable buffer. -means a stranded oligonucleotide. The suitable length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not have to have a sequence completely complementary to a part of the sequence of the template, and it is sufficient that it has sufficient complementarity within a range capable of hybridizing with the template and performing the intrinsic function of the primer. Therefore, the primer in the present invention does not have to have a sequence perfectly complementary to the nucleotide sequence of the gene as a template, and it is sufficient if it has sufficient complementarity within the range in which it can hybridize to this gene sequence and act as a primer. In addition, it is preferable that the primers according to the present invention can be used in a gene amplification reaction.

The amplification reaction refers to a reaction to amplify a nucleic acid molecule, and amplification reactions of such genes are well known in the art, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase These may include lase chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.

In the present invention, the term "probe" means a natural or modified monomer or linear oligomer of linkages, including deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, and means that it exists or is artificially synthesized. The probe according to the present invention may be a single chain, preferably an oligodeoxyribonucleotide. Probes of the present invention may include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probe of the present invention may also contain ribonucleotides. For example, the probes of the present invention can be used with backbone modified nucleotides, such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365:566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexyl tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Tityl-, propynyl-, alkynyl-, thiazoyl-, imidazoyl-, pyridyl- included), 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazoyl-, imidazoyl-, pyridyl-), inosine and diaminopurine .

In addition, the present invention provides a kit for diagnosing Alzheimer's disease comprising the marker for diagnosing Alzheimer's disease or the composition for diagnosing Alzheimer's disease according to the present invention.

The kit for diagnosing Alzheimer's disease of the present invention may include primers or probes capable of measuring the expression level of the biomarker gene of the present invention, the definitions of which are as described above.

If the kit of the present invention is applied to a PCR amplification process, the kit of the present invention optionally includes reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), thermostable DNA polymerase obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs.

Furthermore, the kit according to the present invention may be manufactured in a plurality of separate packaging or compartments including reagent components, and the kit according to the present invention may be a diagnostic kit including essential elements required to perform a DNA chip.

A DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, reagents, enzymes, and the like for producing a fluorescently labeled probe. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

In addition, the present invention, (a) measuring the expression level of the CDK11b, CDK2, CDK5rap3 or CDK7 gene from a biological sample of a patient suspected of having Alzheimer's disease; and (b) comparing the expression level of the gene with the expression level of the corresponding gene in a normal control sample.

The method for measuring the expression level of the gene above may include a known process of isolating RNA from a biological sample using a known technique.

In the present invention, the "biological sample" refers to a sample collected from a living body in which the expression level of the gene according to the degree of occurrence or progression of Alzheimer's disease is different from that of the normal control group, and the sample is, for example, but not limited thereto. However, cells, blood, serum, plasma, saliva, tissue, urine, etc. may be included, and may preferably be plasma.

The expression level of the gene is measured by measuring the level of RNA, preferably the level of mRNA, and methods for measuring the level of mRNA include reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, Multiplex polymerase chain reaction, RNase protection assay, Northern blot and DNA chip, etc., but are not limited thereto.

Therefore, the present invention can confirm the expression level of the marker gene in the control group and the expression level of the marker gene in the Alzheimer's disease patient or suspected patient through the detection methods as described above, and by comparing the level of the expression level with the control group, the Alzheimer's disease It is possible to predict and diagnose the onset, progression or prognosis.

More specifically, as a result of measuring the mRNA level of the marker gene of the present invention, when the mRNA level of the gene is increased compared to a normal control group, it can be determined that Alzheimer's disease has occurred.

Furthermore, the present invention can provide a method for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for the diagnosis of Alzheimer's disease.

The method includes (a) extracting genomic DNA from a biological sample of a patient suspected of having Alzheimer's disease; and (b) using the extracted genomic DNA as a template and performing a multiplex polymerase chain reaction using all of the primer sets consisting of SEQ ID NOs: 5 to 12.

At this time, as a sample for the multiplex polymerase chain reaction, genomic DNA extracted from a biological sample of a patient suspected of having Alzheimer's disease may be used, or DN synthesized through RT-PCR based on RNA extracted from the sample may be used there is.

When the preparation of the template DNA for the multiplex polymerase chain reaction is completed, then all primer sets consisting of SEQ ID NOs: 5 to 12 designed in the present invention are added and a buffer solution for the PCR reaction is added to perform the multiplex polymerase chain reaction. Do it.

In this case, when the primer sets of the present invention are used, the biomarkers of the present invention, CDK11b, CDK2, CDK5rap3 and CDK7, can be rapidly and accurately detected simultaneously through a single multiplex polymerase chain reaction.

Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Discovery of biomarkers for diagnosis of Alzheimer's disease

<1-1> Isolation of plasma and total RNA from the blood of subjects with Alzheimer's disease

In order to discover a new biomarker that can easily diagnose the onset of Alzheimer's disease, the present inventors extracted blood from a 12-week-old 5XFDA mouse, an animal model induced with Alzheimer's disease, and then separated it at 100,000 x g using an ultracentrifuge. Only plasma was separated by centrifugation at 4°C for 1 hour at a speed of . Thereafter, total RNA was extracted by the Trizol method to extract total RNA in the obtained plasma, and the following process was additionally performed to obtain highly pure RNA. Trizol reagent was added at the same volume ratio (1:1) as the volume of the obtained plasma, and then carefully mixed with a micro pipette, followed by incubation at room temperature for about 1 hour. Immediately thereafter, RNA extraction was performed by adding high-purity chloroform in an amount of about 1/5 of the total volume. In particular, when purifying RNA from plasma, 100% isopropanol was added to precipitate the RNA. This step is an important step to obtain RNA capable of genome analysis from a small amount of plasma, and the precipitation step using isopropanol is performed once. Further repetition was performed for a total of 2 times. The concentration and purity of the purified RNA was measured using a nano drop, and it was confirmed that the 260/280 ratio for the extracted RNA was maintained between 1.8 and 2.0.

<1-2> Genomic analysis of total RNA in plasma and discovery of CDK11b, CDK2, CDK5rap3 and CDK7 markers

In order to identify new genes that are increased in an Alzheimer's disease-dependent manner by using the blood plasma as a sample, a whole genome analysis was performed on the total RNA of the blood plasma from the Alzheimer's disease mouse obtained in <1-1>. To this end, a library for whole-genome analysis was prepared using the TruSeq Stranded Total RNA LT Kit known in the art, and expression changes of IncRNA in the blood-derived plasma were analyzed through Illumina HiSeq 4000 Next Generation sequencing analysis, As a result, it was confirmed that the expression of four genes, CDK11b, CDK2, CDK5rap3, and CDK7, was markedly increased in the Alzheimer's disease-induced group compared to the normal control group.

Accordingly, the present inventors confirmed the expression of differentially expressed genes for these four types of genes by calculating (FPKM value, Fragments Per Kilibase of transcript per Million).

As a result, as shown in Figure 1, the RNA amounts of CDK11b, CDK2, CDK5rap3 and CDK7 in the plasma of the Alzheimer's disease group were significantly increased by 317%, 353%, 334% and 320%, respectively, compared to the normal control group confirmed.

Accordingly, the present inventors found that the CDK11b, CDK2, CDK5rap3, and CDK7 genes discovered through the above process could be used as new biomarkers for the diagnosis of Alzheimer's disease.

<Example 2>

Preparation of primers for detecting biomarkers for diagnosis of Alzheimer's disease of the present invention

A primer set that can easily detect the CDK11b, CDK2, CDK5rap3, and CDK7 genes newly discovered as diagnostic markers for Alzheimer's disease in <Example 1> was prepared. For each RNA, cDNA was synthesized using reverse transcriptase, and RT-PCR was performed using the synthesized cDNA as a template to amplify the cDNA of these four genes.

Then, based on the nucleotide sequences of the synthesized CDK11b, CDK2, CDK5rap3, and CDK7 genes, a set of primers in the region that can specifically detect the genes was designed as follows, and detection of the corresponding genes that can be detected using these primers The nucleotide sequence of the region (amplification region) is shown in Table 1 below. On the other hand, the primer set designed in Table 1 below was designed to simultaneously detect these four genes in one PCR reaction. In particular, the primers devised in the present invention described in Table 1 below are designed to enable simultaneous detection of four genes with a single PCR, and the specific region of each gene where the expression level of the four genes is most strongly expressed is selected from the genome. It was confirmed based on analysis, and it was designed so that four genes in the corresponding region have different sizes at the same time and can be accurately detected with high sensitivity and specificity in one PCR reaction.

<Example 3>

Confirmation of detection of biomarkers using the primers of the present invention

The present inventors used the biomarker detection primer sets designed in Example 2 to confirm that CDK11b, CDK2, CDK5rap3 and CDK7 genes, which are increased in the amount of RNA in plasma during the onset of Alzheimer's disease, can be properly detected. Singular RT-PCR was performed on plasma samples from mice with Alzheimer's disease, using the primer sets in Table 1 above. That is, when using a primer set for detecting each corresponding gene, it was analyzed whether the corresponding gene could be detected.

CDK11b singular RT-PCR reaction conditions CDK11b Singular-PCR Reaction Conditions ① Initial denaturation 98℃ / 30sec ② Denaturation 98℃ / 10sec ③ Annealing 64℃ / 30sec --> Perform 40 cycles ④ Extension - 72℃ / 6sec Reaction solution composition Final Primer Concentration 0.5uM dNTP final concentration 0.2 mM Reaction volume: 50ul 50ul template DNA concentration 100 ng

CDK2 singular RT-PCR reaction conditions CDK2 Singular-PCR Reaction Conditions ① Initial denaturation 98℃ / 30sec ② Denaturation 98℃ / 10sec ③ Annealing 64℃ / 30sec --> Perform 40 cycles ④ Extension - 72℃ / 4sec Reaction solution composition Final Primer Concentration 0.5uM dNTP final concentration 0.2 mM Reaction volume: 50ul 50ul template DNA concentration 100 ng

CDK7 singular RT-PCR reaction conditions CDK7 Singular-PCR Reaction Conditions ① Initial denaturation 98℃ / 30sec ② Denaturation 98℃ / 10sec ③ Annealing 64℃ / 30sec --> Perform 40 cycles ④ Extension - 72℃ / 3sec Reaction solution composition Final Primer Concentration 0.5uM dNTP final concentration 0.2 mM Reaction volume: 50ul 50ul template DNA concentration 100 ng

CDK5rap3 singular RT-PCR reaction conditions CDK5rap3 Singular-PCR Reaction Conditions ① Initial denaturation 98℃ / 30sec ② Denaturation 98℃ / 10sec ③ Annealing 64℃ / 30sec --> Perform 40 cycles ④ Extension - 72℃ / 2sec Reaction solution composition Final Primer Concentration 0.5uM dNTP final concentration 0.2 mM Reaction volume: 50ul 50ul template DNA concentration 100 ng

As a result, as shown in FIG. 2, it was found that each of the primer sets designed in the present invention could clearly detect the corresponding genes from the Alzheimer's disease-induced plasma sample.

<Example 4>

Confirmation of simultaneous detection of the biomarkers of the present invention by performing Multiplex-PCR using the primers of the present invention

Furthermore, the present inventors established Multiplex-PCR conditions that can simultaneously detect the four types of biomarkers discovered in the present invention through a single PCR reaction using all eight primers designed in Example 2. By performing the multiplex-PCR reaction conditions in Table 6, it was confirmed through 2% agarose gel electrophoresis after PCR reaction whether CDK11b, CDK2, CDK5rap3, and CDK7 genes could be simultaneously detected. In addition, the present inventors named the following Multiplex-PCR method as AdPlex PCR. In addition, the final concentration of the primer is the concentration used for each primer. On the other hand, if multiplex-PCR is performed under conditions outside of the conditions in Table 6 below, accurate amplification products for the four genes cannot be obtained, and amplification reactions may not occur for some genes, and non-specific amplification products are generated. is difficult to accurately detect. Therefore, Table 6 below can be said to be the optimal reaction conditions for simultaneously detecting 4 types of biomarker genes while using all 8 primers of the present invention.

Multiplex-PCR conditions Multiplex-PCR reaction conditions ① Initial denaturation 98℃ / 30sec ② Denaturation 98℃ / 10sec ③ Annealing 64℃ / 30sec --> Perform 40 cycles ④ Extension - 72℃ / 6sec Reaction solution composition Final Primer Concentration 0.5uM dNTP final concentration 0.2 mM Reaction volume: 50ul 50ul template DNA concentration 100 ng

As a result, as shown in FIG. 5, all of the four CDK11b, CDK2, CDK5rap3, and CDK7 biomarkers can be rapidly and accurately detected from plasma samples through a single PCR reaction after adding all the primer sets designed in the present invention. confirmed that it can.

Therefore, through these experimental results, the present inventors found that CDK11b, CDK2, CDK5rap3, and CDK7 markers could be used as new biomarkers for rapid and accurate diagnosis of Alzheimer's disease. In particular, as it is shown to be increased in plasma, diagnosis can be made easily with only plasma samples, and furthermore, a total of 8 primers designed in the present invention can clearly and simultaneously detect target genes without noise with just one Multiplex-PCR reaction. knew it was possible.

Therefore, the four types of biomarkers discovered in the present invention can be used as new biomarkers for the diagnosis of Alzheimer's disease, and the primer sets according to the present invention can simultaneously detect four types of biomarkers of the present invention with just one PCR reaction. Since it can be rapidly detected, the onset of Alzheimer's can be diagnosed more quickly and accurately.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> Korea Brain Research Institute <120> Novel biomarker composition for diagnosis of Alzheimer's Disease based on multiplex PCR platform <130> NPDC-88356 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 2355 <212> DNA <213> artificial sequence <220> <223> Cdk11b cDNA sequence <400> 1 atgggtgatg aaaaggactc ttggaaagtg aaaacgttag atgaaattct ccaggaaaag 60 aaacgaagga aagaacaaga ggagaaagca gagataaaac gcttaaaaaa ttctgatgac 120 cgcgattcca aaagggattc ccttgaggag ggggagctga gagatcaccg aatggagatc 180 acaatcagga actcaccata tagaagagag gattctatgg aagacagagg agaggaggat 240 gattctctgg ccatcaaacc accccagcaa atgtctcgga aagaaaaggc tcatcacaga 300 aaagacgaga aaagaaaaga gaaacgtcga catcgtagcc attcagcaga gggagggaaa 360 catgccagag tgaaagagaa agaaagggag cacgaacgcc ggaaacgcca ccgagaagaa 420 caagataaag ctcgaaggga gtgggaaaga cagaagagga gggaaatggc gagagaacat 480 tccagaagag agagggaccg cctggagcag ttagaaagga agagggagcg ggagcgcaag 540 ctgagggagc agcagaagga gcagcggggag cagaaggagc gggaacggag ggcagaggag 600 cgccgcaaag agagagaagc gcgtaggggaa gtctctgcac atcaccgtac catgagggag 660 gagtacagtg ataaggggaa ggttggccac tggagccgca gccctctgag gccaccaaga 720 gagcgctttg agatgggaga caaccggaag ccagtaaaag aagagaaggt ggaagagaga 780 gacttgttgt cagacctcca agacatcagt gacagcgaga ggaaaaccag ctcagctgag 840 tcttcatcag cagaatcagg ctcaggttct gaagaggagg aggaggaaga agaagaggaa 900 gaagaagaag aagggagcac cagtgaagaa tcagaggaag aagaggaaga agaagaggag 960 gaggaagaag aggagactgg gagcaactct gaggaggcct ctgaacagtc cgcagaagaa 1020 gtcagtgatg aggaaatgag tgaagatgaa gacagagaaa acgagaacca catcttggtt 1080 gttccagagt cacgatttga ccgagattct ggggacagtg aagaagggga ggaagaagtt 1140 ggtgaaggga ctccacagag cagtgccccc accgaaggag actatgttcc tgactctcca 1200 gcactgtcac ctattgagct aaaacaagaa ctgcccaaat acctgccagc cctgcaggga 1260 tgcaggagtg tagaggagtt tcagtgtctg aacaggattg aagaaggcac ctatggggtg 1320 gtctacagag caaaggacaa gaaaacagat gaaattgtgg ctctgaagcg gttaaagatg 1380 gagaaggaga aggaaggctt cccaatcacg tcgctgaggg aaatcaacac catcctcaag 1440 gcccagcacc ccaacatcgt caccgtcagg gaaattgttg tgggaagtaa catggacaag 1500 atctacattg tgatgaacta cgtggaacat gacctcaaga gcctaatgga gaccatgaag 1560 cagcccttcc tgccagggga ggtgaagacc ctgatgattc agctgctgag tggggtaaag 1620 cacctccatg acaattggat cctacaccgt gacctgaaga cctctaacct cctgctgagc 1680 catgctggca ttctcaaggt gggcgacttt gggctggctc gggaggtatgg ttcaccccta 1740 aaggcctaca ctccagttgt tgtaaccctg tggtatcgtg ccccagaact actgcttggt 1800 gctaaggaat actccacagc tgtggacatg tggtcggtag gctgcatatt tggagaactg 1860 ctgacacaga aacctctgtt ccctgggaag tcagatattg atcagattaa caagattttc 1920 aaggacctgg gtactcctag tgagaaaatc tggcctggct ataatgacct cccagccgtc 1980 aagaagatga ccttcagcga gtatccctat aacaacctcc gcaagagatt tggggctttg 2040 ttatcagatc aaggctttga tctcatgaac aagttcctga catactaccc tggcaggagg 2100 atcaacgcag aagatggcct caagcacgaa tatttccgag agactcccct ccccatcgac 2160 ccatccatgt tccccacgtg gcctgccaag agtgagcagc agagagtgaa gcgaggcacg 2220 agtccacggc ctcctgaggg cggcctgggc tacagccagc tgggtgatga tgacctgaag 2280 gagacgggct tccacctcac caccaccaac cagggagcct cagctgcagg ccctggcttc 2340 agcctcaagt tctga 2355 <210> 2 <211> 897 <212> DNA <213> artificial sequence <220> <223> Cdk2 cDNA sequence <400> 2 atggagaact tccaaaaggt ggagaagatt ggagagggca cgtacggagt ggtgtacaaa 60 gccaaaaaca agttgacggg agaagttggg gcgcttaaga agatccggct cgacactgag 120 actgaaggtg tacccagtac tgccatccga gagatctctc tccttaagga acttaatcac 180 cctaatatcg tcaagctgct ggatgtcatc cacacagaaa ataagcttta tctggttttt 240 gaatttctgc accaggacct caagaaattc atggatgcct ctgctctcac gggcattcct 300 cttcccctca tcaagagcta tctgttccag ctgctccagg gcctggcttt ctgccattct 360 caccgtgtcc ttcaccgaga ccttaagccc cagaacctgc ttatcaatgc agaggggtcc 420 atcaagctgg cagactttgg actagcaaga gcctttggag tccctgtccg aacttacact 480 catgaggtgg tgaccctgtg gtaccgagca cctgaaattc ttctgggctg caagtactac 540 tccacagccg tggatatctg gagcctgggc tgcatctttg ctgaaatggt gacccgcagg 600 gccctattcc ctggagattc tgagattgac caactcttcc ggatctttcg gactctgggg 660 accccagatg aggtggtttg gccaggagtt acttctatgc ctgattataa gccaagtttc 720 cccaagtggg ctcggcaaga ttttagcaaa gttgtgcctc ccctggatga agatggacgg 780 agcttgttat cgcaaatgct gcactatgac cccaacaagc ggatttcagc caaagcagcc 840 ctggctcacc ctttcttcca ggatgtaact aaaccagtgc cccaccttcg gctctga 897 <210> 3 <211> 1512 <212> DNA <213> artificial sequence <220> <223> Cdk5rap3 cDNA sequence <400> 3 atgcaggacc atcagcacgt gcccatcgac atccagacca gcaagctgct cgactggctg 60 gtggacagaa gacactgtaa cctaaaatgg cagagcctgg tgctgaccat ccgggaaaaa 120 atcaacactg ccatccagga catgccagag agccaggaga ttgcccagct gctctctgga 180 tcctacattc attacttcca ctgcctaaga atagtggacc ttcttaaagg caccgaggcg 240 tccaccaaaa atatttttgg ccggtactct tcacaacgga tgaaagattg gcaggagatc 300 gtaagcctgt atgagaagga caacacctat ttagtggaac tctgtagcct cctggttcgg 360 aatgtcagct atgagatccc ctcgctgaag aagcagattg ccaagtgcca gcaactgcag 420 caagaataca gccgcaagga ggaggagggc caggctgggg ccgctgagat gcgagagcaa 480 ttctaccact cctgcaaaca gtatggcatc acgggagaca atgtccgaag agaacttctg 540 gccctggtga aggacctgcc aagtcagctg gctgagatag gggcaggggc tcagtccctg 600 gggggaggcca ttgacctgta ccaggcctgt gtggagtttg tgtgtgacag ccccacagag 660 caagtgctgc ccatgctgcg gtatgtgcag aagaaggggaa actcgacggt gtatgagtgg 720 aggacgggga cagagccctc tgtggtggag cgaccacagc tggaggagcc tcctgagcag 780 gtgcaagagg atgagatcga ctggggtgac tttggggtgg aagctgtttc cgactctggc 840 atcgttgctg agactcctgg aatagactgg ggtatctccc tggagtcaga ggccaaggat 900 gctggggctg acaagataga ctggggtgac gatgctgctg ctgcttcaga gatcaccgtg 960 ctggagacag gaactgaggc tccggagggt gtagctcggg gctcagacgc tctgactctt 1020 ctggaatacc ctgaaactcg gaatcagttc attgatgagc tcatggagct tgagatcttc 1080 ttgtctcaga gagcagtaga gatgagcgag gaggccgaca tcctgtccgt gagccagttc 1140 cagctggctc ctgccatcct ccagggccag accaaagaga agatgctcag cctggtgtcc 1200 acgctgcagc agctgatcgg ccggctcacc agtctgcgga tgcagcacct gtttatgatt 1260 ctggcctcgc caaggtatgt ggaccgagtg acagagttcc tccagcagaa gctgaagcag 1320 tcgcagctgt tggctttgaa gaaggagctg atggtggaga agcagcagga ggcgcttcag 1380 gagcaggcag ctctggagcc caagctggac ctgctgctgg agaagaccag agagctgcag 1440 aaactgattg aagccgacat ctccaagaga tacagtggcc gtcctgtgaa cctgatgggg 1500 acctctctgt ga 1512 <210> 4 <211> 1041 <212> DNA <213> artificial sequence <220> <223> Cdk7 cDNA sequence <400> 4 atggctgtgg acgtgaagtc tcgagcgaag cgctatgaga aactggactt cctcggagag 60 ggacagtttg cgacggtcta taaggccagg gacaagaaca ccaaccaaat cgtcgccatt 120 aagaaaatca aacttggaca caggtcagaa gctaaggatg gaataaatag aacagcctta 180 agggagataa agctcttaca ggaactaagt catccaaata taattggtct ccttgatgca 240 tttggacata agtctaacat tagccttgtc tttgatttta tggaaactga cctcgaggtt 300 attataaagg ataacagcct tgtgctgaca ccatcccaca ttaaagccta catgttgatg 360 actcttcagg gcttagaata tttacatcag cattggattc tacacaggga tctgaaacca 420 aacaacttgt tgttagatga gaatggagtt ctgaaactgg cagattttgg cctggccaaa 480 tcatttggga gccccaatag ggcttacaca catcaagttg tgaccagatg gtaccgggct 540 cctgagttat tgtttggagc taggatgtat ggtgtggggag tagacatgtg ggctgttggt 600 tgtatattag cagaattgct tctaagggtt ccatttttgc ctggagattc agatcttgat 660 cagctaacaa ggatatttga aactctgggt acaccaactg aagagcagtg gcctgacatg 720 tgtagtcttc ccgattatgt gacatttaag agtttccctg gggtcccact gcagcacatc 780 ttcatcgcgg ctggggacga cctgctggag ctcatccaag gcctgttctt atttaacccg 840 tgtacgcgga ccacagcctc acaggcattg aagaccaagt acttcagtaa ccgccccggg 900 ccaacacctg gatgccagct cccgcgacca aactgtccgg tggaggcatt aaaggaaccg 960 gcgaatccaa ccgtggcaac aaagcggaaa agagcagagg ccttagaaca aggaatattg 1020 cccaagaagc tcatttttta g 1041 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220> <223> CDK11b (F) primer sequence <400> 5 gtcagtgatg aggaaatgag t 21 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> CDK11b (R) primer sequence <400> 6 ctcagcgacg tgattgggaa 20 <210> 7 <211> 19 <212> DNA <213> artificial sequence <220> <223> CDK2 (F) primer sequence <400> 7 cagcaaagat gcagcccag 19 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> CDK2 (R) primer sequence <400> 8 ctatctgttc cagctgctcc 20 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> CDK5rap3 (F) primer sequence <400> 9 ctatctgttc cagctgctcc 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> CDK5rap3 (R) primer sequence <400> 10 ggtatgtgca gaagaaggga 20 <210> 11 <211> 19 <212> DNA <213> artificial sequence <220> <223> CDK7 (F) primer sequence <400> 11 cagcccacat gtctactcc 19 <210> 12 <211> 22 <212> DNA <213> artificial sequence <220> <223> CDK7 (R) primer sequence <400> 12 ccaaacaact tgttgttaga tg 22 <210> 13 <211> 398 <212> DNA <213> artificial sequence <220> <223> CDK11b (PCR amplification product) <400> 13 gtcagtgatg aggaaatgag tgaagatgaa gacagagaaa acgagaacca catcttggtt 60 gttccagagt cacgatttga ccgagattct ggggacagtg aagaagggga ggaagaagtt 120 ggtgaaggga ctccacagag cagtgccccc accgaaggag actatgttcc tgactctcca 180 gcactgtcac ctattgagct aaaacaagaa ctgcccaaat acctgccagc cctgcaggga 240 tgcaggagtg tagaggagtt tcagtgtctg aacaggattg aagaaggcac ctatggggtg 300 gtctacagag caaaggacaa gaaaacagat gaaattgtgg ctctgaagcg gttaaagatg 360 gagaaggaga aggaaggctt cccaatcacg tcgctgag 398 <210> 14 <211> 266 <212> DNA <213> artificial sequence <220> <223> CDK2 (PCR amplification product) <400> 14 cagcaaagat gcagcccagg ctccagatat ccacggctgt ggagtagtac ttgcagccca 60 gaagaatttc aggtgctcgg taccacaggg tcaccacctc atgagtgtaa gttcggacag 120 ggactccaaa ggctcttgct agtccaaagt ctgccagctt gatggacccc tctgcattga 180 taagcaggtt ctggggctta aggtctcggt gaaggacacg gtgagaatgg cagaaagcca 240 ggccctggag cagctggaac agatag 266 <210> 15 <211> 114 <212> DNA <213> artificial sequence <220> <223> CDK5rap3 (PCR amplification product) <400> 15 catcctcttg cacctgctca ggaggctcct ccagctgtgg tcgctccacc acagagggct 60 ctgtccccgt cctccactca tacaccgtcg agtttccctt cttctgcaca tacc 114 <210> 16 <211> 178 <212> DNA <213> artificial sequence <220> <223> CDK7 (PCR amplification product) <400> 16 cagcccacat gtctactccc acaccataca tcctagctcc aaacaataac tcaggagccc 60 ggtaccatct ggtcacaact tgatgtgtgt aagccctatt ggggctccca aatgatttgg 120 ccaggccaaa atctgccagt ttcagaactc cattctcatc taacaacaag ttgtttgg 178

Claims (11)

A composition for diagnostic markers for Alzheimer's disease containing all of CDK11b, CDK5rap3 and CDK7 genes. According to claim 1,
The CDK11b is composed of the nucleotide sequence of SEQ ID NO: 1,
The CDK5rap3 is composed of the nucleotide sequence of SEQ ID NO: 3,
The CDK7 is a composition for diagnosis of Alzheimer's disease, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 4. A composition for diagnosing Alzheimer's disease containing all substances measuring the expression levels of CDK11b, CDK2, CDK5rap3 and CDK7 genes,
The material consists of a primer set consisting of SEQ ID NOs: 5 and 6 that specifically bind to CDK11b, a primer set consisting of SEQ ID NOs: 7 and 8 that specifically binds to CDK2, and SEQ ID NOs: 9 and 10 that specifically bind to CDK5rap3. It includes both a primer set consisting of a primer set and a primer set consisting of SEQ ID NOs: 11 and 12 that specifically bind to CDK7, and the CDK11b, CDK2, CDK5rap3 and CDK7 genes can be simultaneously detected in one polymerase chain reaction. To, a composition for diagnosing Alzheimer's disease. delete delete A primer set for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for diagnosis of Alzheimer's disease consisting of SEQ ID NOs: 5 to 12. A kit for diagnosing Alzheimer's disease, comprising the primer set of claim 6. delete delete delete (a) extracting genomic DNA from a biological sample of a patient suspected of having Alzheimer's disease; and
(b) using the extracted genomic DNA as a template and performing a multiplex polymerase chain reaction using all of the primer sets consisting of SEQ ID NOs: 5 to 12;
Method for simultaneous detection of CDK11b, CDK2, CDK5rap3 and CDK7 for diagnosis of Alzheimer's disease.

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