KR100437625B1 - Method for Dectecting the Single Nucleotide Polymorphism by Zip-Code Oligonucleotide Chip and Detection kit - Google Patents

Abstract

According to the present invention, a polymerase chain reaction (PCR) method, an oligonucleotide ligation assay method, a zip-code oligobase attached to a part of the genome sequence of Mycobacterium tuberculosis is applied. Capture consisting of a combination of zip-code oligobase chips, cZip-Code oligobases complementary to Zip-Code oligobases, cZip-Code oligobases and gene sequences to test for single nucleotide polymorphisms (SNPs) The invention relates to a method for testing SNPs using a probe and a reporter probe used to read a type of SNPs corresponding to a nucleotide sequence of a gene to be tested, and a kit used therefor. SNPs can be tested quickly, accurately and in large quantities at the same time.

Description

Method for Dectecting the Single Nucleotide Polymorphism by Zip-Code Oligonucleotide Chip and Detection kit}

The present invention relates to a method for diagnosing Single Nucleotide Polymorphism (SNPs) and a kit used therefor, in particular, a Polymerase Chain Reaction (PCR) method and an Oligonucleotide Ligation Assay method, Zip-Code Oligobase Chip, Complementary Zip-Code (cZip-Code) Oligobase, and Zip-Code Oligobase with Partial Application of Genome Sequence of Mycobacterium tuberculosis Capture probe consisting of cZip-Code oligobase attached to chip and base sequence of gene to test SNPs, reporter used to read kinds of SNPs corresponding to base sequence of gene to test SNPs The present invention relates to a method for testing SNPs using a reporter probe and the like and a kit used therefor.

That is, the present invention includes a PCR method, an oligobase linkage analysis method, and a method for testing SNPs genotype using a Zip-Code oligobase chip, which is applied to a part of the genome sequence of Mycobacterium tuberculosis, and a reagent for the test.

SNPs are a phenomenon in which the bases that make up the genome are partially different from person to person, and human SNPs are estimated to have 0.1% of the human genome composed of about 3 billion base pairs, or about 3 million, which is about 1 per 1,000 bases. (Cooper, DN et al., Hum. Genet. 1985, 69: 201-205).

SNPs are the most common form of DNA sequencing, and mutations that occur in the sequencing of the encoded region (the region that makes the protein from the gene), especially in humans, include cancer, hypertension, diabetes, and dementia. It is reported to be closely related (Sherrington, R. et al., Nature. 1995, 375; 754-760, Kunz R. etal., Hypertension. 1997, 30: 1331-1337, Rogaev, EI et al., Genomics. 1997, 40: 415-424. Niederacher, D. Oncology. 1999, 56: 59-65, Ghaderi, A. Cancer Letters, 2001, 165; 87-94).

Thus, when comparing SNPs between the normal population and the patient population, if specific SNPs appear to be in linkage disequilibrium, these SNPs can be used as very useful genetic markers for specific diseases. (Lai, E., Genomics. 1998, 54: 31-38, Emahazion, T. Trends in Genetics. 2001, 17: 407-413).

Conventional methods for testing SNPs mainly include polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, denaturing gradient gel electrophoresis. Et al. (Cotton, R. Trends Genet. 1997; 13; 43-46). However, these methods are labor-intensive, time-consuming, and have the risk of using radioisotopes and cancer triggers as reagents, making them difficult to use in routine testing methods.

DNA chips are a recently developed method of gene discovery, incorporating mechanical and electronic technologies with existing molecular biology knowledge. A DNA chip is a high-density deposit of hundreds to hundreds of thousands of DNA on a sheet of glass using mechanical automation and electronic control technology. The DNA chip can supplement the above problems of the conventional human SNPs test method, and the biggest difference from the existing test method is that it can easily search for at least several hundred genes at the same time in a short time.

The present invention utilizes the above-described DNA chip technology to supplement the problems of the conventional SNPs test method, and also maximizes the sensitivity and specificity of the test method as well as the advantages of the DNA chip technology.

It is an object of the present invention to provide a method that allows for rapid and accurate SNPs testing at the same time.

Another object of the present invention is to provide a kit comprising a Zip-Code oligobase chip used in the method.

1 is a photograph of a zip-code oligobase chip for monobasic polymorphism test

Figure 2 is a photograph showing the components constituting the test method and test kit using a Zip-Code oligobase chip.

Figure 3 is a PCR reagent picture for monobasic polymorphism test. From left, 10X PCR buffer, Taq DNA Polymerase, and 10 mM dNTPs solution are shown.

Figure 4 is a photograph of oligobase linkage assay reagent for monobasic polymorphism test. From left, 10X Ligation buffer, Taq DNA Ligase, capture news, Cy 5 reporter news, and Cy 3 reporter news.

Figure 5 is a photograph of a kit containing 10 Zip-Code oligobase chips for monobasic polymorphism test and the chamber for binding.

In order to achieve the above object, the present invention comprises amplifying the gene to be tested for SNPs by a PCR method, by mixing a reporter probe (reporter probe) that combines the capture signal and the fluorescent material in the amplified product, It provides a method of testing SNPs comprising the step of performing a base linkage assay method, the step of coupling the product of the oligobase linkage assay method to a Zip-Code oligobase chip, and reading the SNPs.

In the present invention, the sequence of the zip-code oligobase to which a part of the genome sequence of Mycobacterium tuberculosis adhered to the zip-code oligobase chip is applied is sequence information 1 to 58, and corresponds to the zip-code oligobase. The sequence of the cZip-Code oligobase is characterized in that SEQ ID NO: 59-116.

In addition, the fluorescent material that can be used in the present invention is Cy2, GFP, YO-PRO-1, YOYO-1, Calcein, FITC, FlourX, ALEXA 488, Rhodamine 110, ABI 5-FAM, Oregon Green 500, Oregon green 488, RiboGreen , Rhodamine Green, Rhodamine 123, Magnesium Green, Calcium Green, TO-PRO-1, TOTO-1, ABI JOE, BODIPY 530/550, Dil, BODIPY TMR, BODIPY 558/568, BODIPY564 / 570, Alexa 546, TRITC, Magnesium Orange, Phycoerythrin R & B, Rhodamine Phalloidin, Calcium Orange, Pyronin Y, Rhodamine B, ABI TAMRA, Rhodamine Red, Cy3.5, ABI ROX, Calcium Crimson, Alexa 594, Texas Red, Nile Red, YO-PRO-3 , YOYO-3, R-phycocyanin, C-phycocyanin, TO-PRO-3, TOTO-3, DiD DilC (5), Thiadicarbocyainie, Cy5.5 or Cy5 and Cy3 are preferred, and more preferably other It is preferable to use Cy5 and Cy3 because the fluorescent structure itself of Cy5 and Cy3 is relatively stable than the fluorescent material, and the non-specific fluorescent reaction is less than that of other fluorescent materials.

The present invention also provides a kit for testing SNPs comprising a) a reagent for PCR of a gene to be tested for SNPs, b) a reagent required for an oligobase linkage analysis method, and c) a Zip-Code oligobase chip for testing SNPs.

Hereinafter, the present invention will be described in detail.

The composition of the present invention can be divided into three parts. First, a process of specifically amplifying a nucleotide sequence region of a gene to be tested for SNPs from a genome using a PCR method, and a second oligobase linkage using Taq DNA ligase. The analysis process, and the third step is to prepare a Zip-code oligobase chip for testing SNPs to read the SNPs.

In addition, we developed a Zip-Code oligobase chip test kit for testing SNPs to facilitate the use of reagents and instruments for each process.

SNPs test method using the present invention Zip-Code oligobase chip is composed of the following techniques.

1) After amplification by PCR process upstream and downstream primers, buffers and other reagents corresponding to the nucleotide sequence of the gene to be examined DNA and SNPs,

2) After the oligobase linkage assay (see Examples 3, 5 and 7) was performed by mixing the product amplified by PCR with the reporter reporter, the capture reporter, the buffer solution, and the Taq DNA ligase,

3) Zip-code oligobase chip is produced by synthesizing amine group (-NH2) to be bonded to 5 'end of Zip-Code oligobase,

4) Using a microarray device, the Zip-Code oligobase is attached to the glass plate for chip making at regular intervals and arrangement to prepare a Zip-Code oligobase chip, followed by washing and fixing.

5) Here, the product of the oligobase linkage analysis method of 2) is hybridized at a constant temperature, and then washed to remove non-specifically bound ones.

6) SNPs inspection method using Zip-Code oligobase chip, which consists of reading SNPs with a chip scanner.

The type and base sequence structure of the Zip-Code oligobase are shown in Table 1, and the base sequence of the cZIp-Code oligobase is shown in Table 2.

[Table 1] Types and base sequence structure of Zip-Code oligobase

Kinds Zip-Code Oligobase Structure TBzip 01 5 '-NH ₂ C ₆ -tgt gct tac cgc acc tcg cag tcg t-3' TBzip 02 5 '-NH ₂ C ₆ -atc ttg cgc ggc agc tcg tcg acc g-3' TBzip 03 5 '-NH ₂ C ₆ -aaa gcg ggc ggc gat cgc gaa tgt c-3' TBzip 04 5 '-NH ₂ C ₆ -aga ttg gga tgc ggt cgc gat acc g-3' TBzip 05 5 '-NH ₂ C ₆ -gag gat ctg tag cgc ctc ttc gag c-3' TBzip 06 5 '-NH ₂ C ₆ -cga act cga agc cga gct ggc ggt g-3' TBzip 07 5 '-NH ₂ C ₆ -gtg gtc cat cac aaa cag gga gtc g-3' TBzip 08 5 '-NH ₂ C ₆ -ctt gag cga tga cgg acg gga aaa g-3' TBzip 09 5 '-NH ₂ C ₆ -aag ttg ggg atc tgt aga ccc agc c-3' TBzip 10 5 '-NH ₂ C ₆ -gat cgg ccg gtg aag cga aag gtt c-3' TBzip 11 5 '-NH ₂ C ₆ -gat ggt gat ccc gcg cgt gcc gaa a-3' TBzip 12 5 '-NH ₂ C ₆ -gga ttg cac cgt cag cac cac cga g-3' TBzip 13 5 '-NH ₂ C ₆ -tcc cag gac ggc gct ggc acg ttg a-3' TBzip 14 5 '-NH ₂ C ₆ -cgg cgt cca cgt cga gtt cct tcg c-3' TBzip 15 5 '-NH ₂ C ₆ -tgt gcg ccc gag atc ggt atc ccc g-3' TBzip 16 5 '-NH ₂ C ₆ -atc gca tcg tga tgg cgt aag ctc c-3' TBzip 17 5 '-NH ₂ C ₆ -ttc ggg gaa act ccg cac cgc cac g-3' TBzip 18 5 '-NH ₂ C ₆ -tag gtt tgg cca gtg cgt tgg atc g-3' TBzip 19 5 '-NH ₂ C ₆ -tcg aca acc cgg ttg gag gat tca g-3' TBzip 20 5 '-NH ₂ C ₆ -cca aaa gct tta cgc cag cgc cga a-3'

Table 1. Zip-Code Oligobase Types and Sequence Structures (Continued)

Kinds Zip-Code Oligobase Structure TBzip 21 5 '-NH ₂ C ₆ -ccg tac cct tcc gct gga gat tta c-3' TBzip 22 5 '-NH ₂ C ₆ -aga tcg gtg agc agt tca aag ccg g-3' TBzip 23 5 '-NH ₂ C ₆ -ggg tat ccg ttc ggt gtt gcg tag t-3' TBzip 24 5 '-NH ₂ C ₆ -acc tgg tca atg gga cca ttg gtc c-3' TBzip 25 5 '-NH ₂ C ₆ -tat gtc agt gac gcg ctc agc gtt g-3' TBzip 26 5 '-NH ₂ C ₆ -tgg tgc tgg cgc aga cct ttg tct c-3' TBzip 27 5 '-NH ₂ C ₆ -acc gcg caa atg gac agt gtg gcc a-3 TBzip 28 5 '-NH ₂ C ₆ -gac ccc aac ttg aca cgt cgc aag g-3' TBzip 29 5 '-NH ₂ C ₆ -gga gag ttt ggc gcg acc cta acc t-3' TBzip 30 5 '-NH ₂ C ₆ -tgg cga gag tgt ctc gtc gat cat c-3' TBzip 31 5 '-NH ₂ C ₆ -gtt ggg tat atc tcc cgg cga tcg c-3' TBzip 32 5 '-NH ₂ C ₆ -gta ttg gtg ctc gag tcc ggc acg a-3' TBzip 33 5 '-NH ₂ C ₆ -gtc tac gcc atc gcg gtg cta aag c-3' TBzip 34 5 '-NH ₂ C ₆ -agc atc gca ttc agt acc gcg gct g-3' TBzip 35 5 '-NH ₂ C ₆ -cgt aag cct cgt cag cta tcc ggg g-3' TBzip 36 5 '-NH ₂ C ₆ -cca aac gca ccc caa cct gtc cgg a-3' TBzip 37 5 '-NH ₂ C ₆ -gtc ggg ggt atc gcg ttg ctc tac g-3' TBzip 38 5 '-NH ₂ C ₆ -cgg cgg tgg cat tgt cac tgc tgc t-3 TBzip 39 5 '-NH ₂ C ₆ -tcg aat tct cgg tgt ccg cgg gcg a-3' TBzip 40 5 '-NH ₂ C ₆ -atg cac ccg cct acg gat ctg cct c-3'

TBzip 41 5 '-NH ₂ C ₆ -gac gat tgg cgg cac tgg cta tcg c-3' TBzip 42 5 '-NH ₂ C ₆ -gca gtt cgt ggc cat ggt gac cgc t-3' TBzip 43 5 '-NH ₂ C ₆ -ccg tcg agt cgg gct gct gtt gca a-3' TBzip 44 5 '-NH ₂ C ₆ -ttc gga gcc cgt atc gcc gga gtc a-3' TBzip 45 5 '-NH ₂ C ₆ -tcg gga ccg ata cca ggt agc cgg t-3' TBzip 46 5 '-NH ₂ C ₆ -agt act ggc gtt tgc ccg ctc gct c-3' TBzip 47 5 '-NH ₂ C ₆ -gag tct gac gcc gcc cac ttc gac t-3' TBzip 48 5 '-NH ₂ C ₆ -cgt tgt ggt agc ggc act ggt ggt g-3' TBzip 49 5 '-NH ₂ C ₆ -tcc ttg cgt cgc gcc gtt aac acg g-3' TBzip 50 5 '-NH ₂ C ₆ -gcc tcg ctg cag tct gcg ttc act c-3' TBzip 51 5 '-NH ₂ C ₆ -ctg ctg ttg agc gtg aag acc gcc g-3' TBzip 52 5 '-NH ₂ C ₆ -ggt gct tgc agt gct cgg gcc caa c-3' TBzip 53 5 '-NH ₂ C ₆ -ctg ggt gtg ggt gct cgt acg ccg a-3' TBzip 54 5 '-NH ₂ C ₆ -cga cgc ggg ctt ggt acg ttt ggg g-3' TBzip 55 5 '-NH ₂ C ₆ -gtg gtc tgc cag ccg tcg gtg cca t-3' TBzip 56 5 '-NH ₂ C ₆ -ccg gtc gat cgt ggt gtt cgc ggc t-3' TBzip 57 5 '-NH ₂ C ₆ -cat gag caa gct gca gct gcg cgc g-3' TBzip 58 5 '-NH ₂ C ₆ -gtt acc gct ggt gct gcc gcc ggt a-3'

[Table 2] Types and base sequence structure of cZip-Code oligobase

No Sequence No Sequence 01 5'-acg act gcg agg tgc ggt aag cac a-3 ' 30 5'-gat gat cga cga gac act ctc gcc a-3 ' 02 5'-cgg tcg acg agc tgc cgc gca aga t-3 ' 31 5'-gcg atc gcc ggg aga tat acc caa c-3 ' 03 5'-gac att cgc gat cgc cgc ccg ctt t-3 ' 32 5'-tcg tgc cgg act cga gca cca ata c-3 ' 04 5'-cgg tat cgc gac cgc atc cca atc t-3 ' 33 5'-gct tta gca ccg cga tgg cgt aga c-3 ' 05 5'-gct cga aga ggc gct aca gat cct c-3 ' 34 5'-cag ccg cgg tac tga atg cga tgc t-3 ' 06 5'-cac cgc cag ctc ggc ttc gag ttc g-3 ' 35 5'-ccc cgg ata gct gac gag gct tac g-3 ' 07 5'-cga ctc cct gtt tgt gat gga cca c-3 ' 36 5'-tcc gga cag gtt ggg gtg cgt ttg g-3 ' 08 5'-ctt ttc ccg tcc gtc atc gct caa g-3 ' 37 5'-cgt aga gca acg cga tac ccc cga c-3 ' 09 5'-ggc tgg gtc tac aga tcc cca act t-3 ' 38 5'-agc agc agt gac aat gcc acc gcc g-3 ' 10 5'-gaa cct ttc gct tca ccg gcc gat c-3 ' 39 5'-tcg ccc gcg gac acc gag aat tcg a-3 ' 11 5'-ttt cgg cac gcg cgg gat cac cat c-3 ' 40 5'-gag gca gat ccg tag gcg ggt gca t-3 ' 12 5'-ctc ggt ggt gct gac ggt gca atc c-3 ' 41 5'-gcg ata gcc agt gcc gcc aat cgt c-3 ' 13 5'-tca acg tgc cag cgc cgt cct ggg a-3 ' 42 5'-agc ggt cac cat ggc cac gaa ctg c-3 ' 14 5'-gcg aag gaa ctc gac gtg gac gcc g-3 ' 43 5'-ttg caa cag cag ccc gac tcg acg g-3 ' 15 5'-cgg gga tac cga tct cgg gcg cac a-3 ' 44 5'-tga ctc cgg cga tac ggg ctc cga a-3 ' 16 5'-gga gct tac gcc atc acg atg cga t-3 ' 45 5'-acc ggc tac ctg gta tcg gtc ccg a-3 ' 17 5'-cgt ggc ggt gcg gag ttt ccc cga a-3 ' 46 5'-gag cga gcg ggc aaa cgc cag tac t-3 ' 18 5'-cga tcc aac gca ctg gcc aaa cct a-3 ' 47 5'-agt cga agt ggg cgg cgt cag act c-3 ' 19 5'-ctg aat cct cca acc ggg ttg tcg a-3 ' 48 5'-cac cac cag tgc cgc tac cac aac g-3 ' 20 5'-ttc ggc gct ggc gta aag ctt ttg g-3 ' 49 5'-ccg tgt taa cgg cgc gac gca agg a-3 '

Table 2 Types and Sequences of cZip-Code Oligobases (cont.)

No Sequence No Sequence 21 5'-gta aat ctc cag cgg aag ggt acg g-3 ' 50 5'-gag tga acg cag act gca gcg agg c-3 ' 22 5'-ccg gct ttg aac tgc tca ccg atc t-3 ' 51 5'-cgg cgg tct tca cgc tca aca gca g-3 ' 23 5'-act acg caa cac cga acg gat acc c-3 ' 52 5'-gtt ggg ccc gag cac tgc aag cac c-3 ' 24 5'-gga cca atg gtc cca ttg acc agg t-3 ' 53 5'-tcg gcg tac gag cac cca cac cca g-3 ' 25 5'-caa cgc tga gcg cgt cac tga cat a-3 ' 54 5'-ccc caa acg tac caa gcc cgc gtc g-3 ' 26 5'-gag aca aag gtc tgc gcc agc acc a-3 ' 55 5'-atg gca ccg acg gct ggc aga cca c-3 ' 27 5'-tgg cca cac tgt cca ttt gcg cgg t-3 ' 56 5'-agc cgc gaa cac cac gat cga ccg g-3 ' 28 5'-cct tgc gac gtg tca agt tgg ggt c-3 ' 57 5'-cgc gcg cag ctg cag ctt gct cat g-3 ' 29 5'-agg tta ggg tcg cgc caa act ctc c-3 ' 58 5'-tac cgg cgg cag cac cag cgg taa c-3 '

To prepare the SNPs test kit using the Zip-Code oligobase chip of the present invention, the following reagents, apparatus, and apparatus were configured.

1) For PCR of genes to test SNPs,

10X PCR buffer, 10 mM dNTPs, and Taq DNA polymerase 250 units (Amplitaq, PE, USA)

2) For oligobase linkage analysis to test SNPs,

Attach the fluorescent substance Cy5 (green fluorescent substance) or Cy3 (red fluorescent substance) to the 5 'end of each reporter consisting of oligobases corresponding to the respective SNPs,

Attaching a phosphate group to the 5 'end of the oligobase of each capture sequence consisting of a part of the base sequence of the gene to be tested for SNPs and a cZip-Code oligobase,

10X ligase buffer, and

Taq DNA ligase 40,000 units (NEB, UK)

3) For fabrication of Zip-Code oligobase chips to test SNPs

Attachment of an amine group (-NH2) to the 5 'end of the Zip-Code oligobase,

Microscopic glass slides whose surfaces are coated with aldehyde groups,

Microarray devices (Bio-Robotics, UK),

A hybridization chamber (Sigma, USA) for splitting the glass slide into eight zones, and

0.2% Sodium Dodecyl Sulfate (SDS) Solution, NaBH ₄ (Sigma, USA), Phosphate Buffered Saline (PBS) Solution, 100% Ethanol

4) In order to bind the oligobase linkage analysis product to the Zip-Code oligobase chip,

Zip-Code oligobase chip made in 3)

12X SSPE Hybrid Binding Buffer, and

10% SDS

5) 3X SSPE solution, 1X SSPE solution for washing the combined Zip-Code oligobase chip

6) Microarray scanner (Gsilumonics, USA), etc., for reading Zip-Code oligobase chips.

The SNPs can be tested with the reagents, instruments, and equipment. Based on this, a Zip-Code oligobase chip test kit for testing SNPs was constructed as follows.

1) For PCR of genes to test SNPs,

1.5 ml tube containing 1 ml of 10 mM dNTPs

1.5 ml tube containing 1 ml of 10X PCR buffer and

1.5 ml tube containing Taq DNA polymerase 250 units (Ampli Tag., PE, USA)

2) for oligobase linkage analysis method for testing SNPs,

Reporter reporter 1.5 ml tubes with Cy 5 or Cy 3 attached to the 5 'end, respectively

Capture ml 1.5 ml tube with phosphate attached at 5 'end

1.5 ml tubes of 10X ligase buffer and

Taq DNA ligase 2,000 units (NEB, UK) 1.5 ml tubes

3) In order to hybridize the oligobase linkage analysis product to the Zip-Code oligobase chip,

Zip-Code Oligobase Chip

12X SSPE Hybridization Buffer 1.5 ml Tube

8 well hybridization chamber (Sigma, USA)

After amplifying the gene to be tested for SNPs by PCR using the kit, it takes about 8 hours to test the SNPs with a Zip-Code oligobase chip. In addition, one Zip-Code oligobase chip can be used to test at least several hundred SNPs simultaneously.

Zip-Code oligobase chip kit of the present invention is a method capable of routinely testing SNPs, and is an industrially useful invention.

It will be described in more detail through the following non-limiting examples of the invention.

Example 1 PCR Procedures for Testing SNPs of Human p53 Gene

A buffer solution containing 50 ng of DNA, 200 uM of dNTPs, 10 pmol each of the upstream and downstream primers (Table 3), and Taq DNA polymerase 1.0 unit in a 500 μl PCR tube (50 mM KCl, 10 mM Tris-HCl pH 8.4, 1.5 mM MgCl ₂ , 0.1% Triton X-100) is added and the total volume is 50 μl with distilled water.

B) After centrifugation by mixing the reaction solution, and warmed once at 95 ℃ for 4 minutes in a thermostat (9600 thermal cycler, PE, USA), then 95 ℃ 50 seconds, 58 ℃ 20 seconds, 72 After repeating the procedure of 30 ° C. for 35 seconds, it was warmed once at 72 ° C. for 7 minutes and then stored at 4. The process takes about two and a half hours.

[Table 3] Primer types and sequence structure for PCR of human p53 gene

Primer type Sequence structure Codon 72 Forward 5 '-atc tac agt ccc cct tgc cg-3' Reverse 5 '-gca act gac cgt gca agt ca-3' Codon 205 Forward 5 '-acc atg agc gct gct cag at-3' Reverse 5 '-agt tgc aaa cca gac ctc ag-3' Codon 239 Forward 5 '-gtg tta tct cct agg ttg gc-3' Reverse 5 '-tcc agg tca gga gcc act tg-3'

Example 2: Reporter and capture newsletter fabrication for testing SNPs of human p53 gene

A) Cy5 attachment with green fluorescence at the 5 'end of the reporter reporter for testing normal SNPs of the human p53 gene (Table 4)

B) Cy3 attachment with red fluorescence at the 5 'end of reporter reporter for testing Mutant SNPs of human p53 gene (Table 4)

C) Attaching a phosphate group to the 5 'end of the capture sequence consisting of a portion of the base sequence containing SNPs in the human p53 gene and a cZip-Code oligobase (Table 5)

[Table 4] Type and structure of reporter reporter for SNPs test of human p53 gene

Kinds Sequence structure Codon 72 Cy5 Reporter News 5 '-Cy5-aga ggc tgc tcc ccg-3' Cy3 Reporter News 5 '-Cy3-aga ggc tgc tcc ccc-3' Codon 205 Cy5 Reporter News 5 '-Cy5-gaa gga aat ttg cgt gtg gag tt-3' Cy3 Reporter News 5 '-Cy3-gaa gga aat ttg cgt gtg gag ta-3' Codon 239 Cy5 Reporter News 5 '-Cy5-cca tcc act aca act aca tgt gtg-3' Cy3 Reporter News 5 '-Cy3-cca tcc act aca act aca tgt gta-3'

Table 5 Types and structures of capture news for testing SNPs of human p53 gene

Kinds Sequence structure Codon 72 Capture News 5 '-P-cgt ggc ccc tgc aca cga ctg cga ggt gcg gta agc aca-3' Codon205 Capture News 5 '-P-ttt gga tga cag aaa cac ttt tcg cgg tcg acg agc tgc cgc gca aga t-3' Codon239 caught news 5 '-P-aca gtt cct gca tgg gcg aca ttc gcg atc gcc gcc cgc ttt-3'

Example 3: Oligobase Linkage Assay for Testing SNPs in the p53 Gene

1) 500 pmol PCR tubes with 500 fmol of PCR product, 2 pmol of Cy5 and Cy3 reporter each, 2 pmol of capture signal, and buffer (20 mM Tris-HCl, 25 mM Potassium acetate, 10 mM Magnesium acetate, 10 mM) DTT, 1 mM NAD, 0.1% Triton X-100, pH 7.6)) is added and the total volume is 50 μl with distilled water.

2) After mixing and centrifuging, warm it up to 95 ℃ once for 2 minutes in a thermostat (9600 thermal cycler, PE, USA), and then add tag DNA ligase 40 Units. Repeat the process for 4 minutes 35 times.

Hereinafter, the oligobase linkage analysis method will be described in more detail with reference to a normal SNP among SNPs present in the human p53 gene Exon 4 codon 72.

1) The normal sequence of human p53 gene Exon 4 codon 72 is cgc and the amino acid is Arginine. However, if the cgc sequence is mutated to ccc (g: normal SNP → c: mutant SNP), the amino acid designates Proline, not Arginine.

2) Taq DNA ligase binds each base by combining OH at 3 'end of one base and Phosphate at 5' end of another base as a phosphodiester group. It acts as a linker, and when one of the two bases is not bound to the template, it cannot link to each base.

Principles of Oligobase Link Analysis Methods

500 pmol PCR product of human p53 gene Exon 4 with normal SNP present in Codon 72 according to the oligobase linkage analysis method described above, 2 pmol reporter reporter for normal SNP test with Cy5 attached, mutant SNP test with Cy3 attached After adding 2 pmol of reporter reporter, 2 pmol of Codon 72 capture reporter, and 40 mLs of buffer and Tag DNA ligase to 500 μl PCR tube, perform oligobase linkage analysis as follows.

1) Cy5 is attached to PCR product (template) of human p53 gene Exon 4 with normal SNP

When reporter news and capture news for normal SNP search are combined

The reporter and capture news for normal SNP retrieval with Cy5 attached to the gap linked by Taq DNA ligase are separated from the template by thermal denaturation and form single strands as follows.

2) When the reporter and capture reporter of the mutant SNP searcher with Cy3 attached to the PCR product (template) of the human p53 gene Exon 4 in which the normal SNP is present are combined

Since there is no gap between the reporter and capture reporter for Cy3 attached mutant SNP retrieval, the reporter and capture reporter for Cy3 attached mutant SNP retrieval are separated from the template by thermal denaturation. Are separated.

Example 4: Zip-Code Oligobase Chip Fabrication Method for Examining SNPs of p53 Gene

1) Add 20 µl of 2X spoting solution to 20 µl of each Zip-Code oligobase (40 pmol / µl) and mix.

2) In the microarray apparatus, 58 kinds of Zip-Code oligobases prepared at the concentration of 20 pmol / μl by dividing the microscope glass sliders prepared in the above reagents, instruments and equipment items in each zone were identical in each zone. Attach it.

3) The glass slide to which the Zip-Code oligobase is attached was washed twice with 500 ml of 0.2% SDS solution for 2 minutes and twice with distilled water, followed by 1.3 g NaBH ₄ and 375 ml PBS (Phosphate vuffered saline). ) After standing in 125 ml ethanol mixed solution for 5 minutes, washed again with 0.2% SDS solution 3 times for 1 minute, washed twice with distilled water for 1 minute, and then drained. Zip-Code oligobase chip Used as.

Example 5: Zip-Code Oligobase Chip and Oligobase to Test SNPs of p53 Gene

Comprehensive Bonding of Linked Assay Products

1) The zip-code oligobase chip prepared in Example 4 is brought into close contact with the chamber for 8 wells binding in the reagent and instrument items.

2) Add 10 µl of oligobase linkage assay product, 50 µl of distilled water and 60 µl of 12X SSPE conjugated buffer to 500 µl PCR tube to make the total volume 120 µl, and then add 0.5 µl of 10% SDS solution. Warm for 5 minutes at ℃, and then stand on ice for 3 minutes.

3) After centrifugation, inject 100 µl of the above solution into each well of the Zip-Code oligobase chip, seal the well with a tape to prevent evaporation, and then bind for 3 hours in a thermostat maintained at 42 ° C. Let's do it.

Hereinafter, the fusion process of the zip-code oligobase chip and the oligobase linkage assay product will be described in more detail by taking a normal SNP among the SNPs present in the human p53 gene Exon 4 codon 72 as an example.

1) When oligonucleotide linkage analysis present of normal SNP retrieval reporter and capture reporter with gap linked by Taq DNA ligase is conjugated to Zip-Code oligobase chip

2) Oligobase linkage analysis present of reporter and capture reporter for GNP-linked Cy3 attached mutant SNP search is conjugated to Zip-Code oligobase chip.

Example 6: Washing of Zip-Code Oligobase Chips

1) Put the Zip-Code oligobase chip after the hybridization reaction in a container containing 200 ml of 3X SSPE buffer solution and washed once for 2 minutes.

2) Transfer to a container containing 200ml of 1X SSPE buffer solution and wash once for 2 minutes.

3) After removing water, it was stored in a place where light was blocked until the result was confirmed.

Example 7: Reading of Zip-Code Oligobase Chips

1) Set the scanner to detect the excitation wavelength of 550 nm and the emission wavelength of 570 nm, and then put the washed Zip-Code oligobase chip into the fluorescent materials Cy5 (green fluorescent material) and Cy3 (red fluorescent material). The SNPs in the p53 gene were read by analyzing whether or not the oligobase linkage product and the Zip-Code oligobase chip were conjugated with each other and their positions (Table 6).

Hereinafter, the reading process of the Zip-code oligobase chip will be described in more detail by taking a normal SNP among the SNPs present in the human p53 gene Exon 4 codon 72 as an example.

1) When the oligonucleotide-linked assay product, which has a gap between the reporter for the normal SNP search and the capture for the Cy5 attachment, is conjugated to the Zip-Code oligobase chip

Green fluorescence is indicated by Cy5 located 5 'of the conjugated oligobase linkage assay product at the position to which the TBZip 01 Zip-Code oligobase is attached in the Zip-Code oligobase chip (Table 6).

2) An oligonucleotide linkage assay presenting a gap between Cy3-attached mutant SNP retrieval reporter and capture reporter is conjugated to Zip-Code oligobase chip.

5 'of the oligobase linkage analysis product conjugated to the position where TBZip 01 Zip-Code oligobase is attached in the Zip-Code oligobase chip does not have fluorescence because Cy3, which is a red phosphor, is not attached.

In contrast to the above case, in the case of the mutant SNP, red fluorescence is generated by Cy3 located at 5 'of the oligobase linkage assay product conjugated to the position where TBZip 01 Zip-Code oligobase is attached in the Zip-Code oligobase chip. , Cy5 is not attached, so green fluorescence does not appear (Table 6).

When normal SNPs and mutant SNPs are mixed, Cy5 and Cy3 are attached to the TBZip 01 Zip-Code oligobase attached to the Zip-Code oligobase chip, resulting in yellow fluorescence of green and red complementary colors. Table 6).

Table 6 SNPs read result of human p53 gene by Zip-Code oligonucleotide chip

Codon chip SNPs Results Codon 72 CGC Normal homozygote CGC / CCC Nor / Mut Heterozygote CCC Mutant Homozygote Codon 205 TAT Normal homozygote TTT Mutant Homozygote Codon 239 AAC Normal homozygote GAC Mutant Homozygote

Each round dot in the chip section of Table 6 indicates that the oligobase linkage products are bound to the Zip-Code oligobase in each Zip-Code oligobase chip. Green dots represent normal SNPs and red dots represent mutant SNPs. Yellow dots are indicated by complementary colors of green and red, indicating a mixture of normal and mutant SNPs.

As shown in the above results, kitting Zip-Code oligobase chips and reagents used in the SNPs test method using the Zip-Code oligobase chip of the present invention makes it easy to test SNPs for a large amount of samples without much time and effort. As can be seen, the scope of protection of the present invention includes both the test method and the test kit, and the method of kitting is described in Example 8 below.

Example 8: SNPs test method and kit method using Zip-Code oligobase chip

SNPs test kit using the Zip-Code oligobase chip of the present invention is a kit kit of the reagents involved in the PCR process of the gene to test SNPs, a kit kit of the reagents involved in the oligobase linkage analysis process, And it consists of three parts of the Zip-Code oligobase chip to read and confirm the SNPs.

A) Kit the reagents configured for PCR of the genes to be tested for SNPs

Reagents used for PCR of the genes to be tested for SNPs were kitted as described in Example 1. The configuration consists of a 1.5 ml tube containing 1 ml of 10X PCR buffer, a 1.5 ml tube containing 200 ul of 10 mM dNTPs solution, and a 1.5 ml tube containing 200 units of Taq DNA polymerase (Amplitaq, PE, USA). Consists of. All of these are stored in a -20 ° C. freezer and the method of use is the same as in Example 1.

B) Kit kit for reagents used in oligobase linkage analysis

Reagents used for oligobase linkage assays were kitted as described in Example 3. The configuration consists of a 1.5 ml tube containing 1 ml of 10 × buffer and a tube containing 40,000 units of Tag DNA ligase (NEB, UK). All of these were stored in a -20 ° C. freezer and the method of use was the same as in Example 3.

All). Zip-Code oligobase chip kit

The Zip-Code oligobase chip prepared according to the fabrication method of Example 4 was composed of 10 oligobase chips capable of processing eight specimens in one sheet, and the method of use was the same as in Example 5 (FIG. 5). .

As shown in Table 6, the SNPs of the human p53 gene using the Zip-Code oligobase chip kit for testing the SNPs of the present invention were able to read each SNPs with high sensitivity and specificity, and to accurately read the SNPs. It can be rapidly tested within about 8 hours from the amplification of the sample by PCR process to reading the SNPs with the Zip-Code oligobase chip, indicating that it is an industrially useful method for screening SNPs.

<110> MyGene Ltd.; HAN IN KWON <120> Method for Dectecting the Single Nucleotide Polymorphism by Zip-Code Oligonucleotide Chip and Detection kit <160> 131 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 1 tgtgcttacc gcacctcgca gtcgt 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 2 atcttgcgcg gcagctcgtc gaccg 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 3 aaagcgggcg gcgatcgcga atgtc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 4 agattgggat gcggtcgcga taccg 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 5 gaggatctgt agcgcctctt cgagc 25 <210> 6 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 6 cgaactcgaa gccgagctgg cggtg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400 > 7 gtggtccatc acaaacaggg agtcg 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 8 cttgagcgat gacggacggg aaaag 25 <210> 9 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 9 aagttgggga tctgtagacc cagcc 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 10 gatcggccgg tgaagcgaaa ggttc 25 <210> 11 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 11 gatggtgatc ccgcgcgtgc cgaaa 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400 > 12 ggattgcacc gtcagcacca ccgag 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 13 tcccaggacg gcgctggcac gttga 25 <210> 14 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 14 cggcgtccac gtcgagttcc ttcgc 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 15 tgtgcgcccg agatcggtat ccccg 25 <210> 16 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 16 atcgcatcgt gatggcgtaa gctcc 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400 > 17 ttcggggaaa ctccgcaccg ccacg 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 18 taggtttggc cagtgcgttg gatcg 25 <210> 19 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 19 tcgacaaccc ggttggagga ttcag 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 20 ccaaaagctt tacgccagcg ccgaa 25 <210> 21 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 21 ccgtaccctt ccgctggaga tttac 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence < 400> 22 agatcggtga gcagttcaaa gccgg 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 23 gggtatccgt tcggtgttgc gtagt 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 24 ac ctggtcaa tgggaccatt ggtcc 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 25 tatgtcagtg acgcgctcag cgttg 25 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotde sequence <400> 26 tggtgctggc gcagaccttt gtctc 25 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 27 accgcgcaaa tggacagtgt ggcca 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 28 gaccccaact tgacacgtcg caagg 25 <210> 29 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequ ence <400> 29 ggagagtttg gcgcgaccct aacct 25 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 30 tggcgagagt gtctcgtcga tcatc 25 <210> 31 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 31 gttgggtata tctcccggcg atcgc 25 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> < 223> Zip code oligonucleotide sequence <400> 32 gtattggtgc tcgagtccgg cacga 25 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 33 gtctacgcca tcgcggtgct aaagc 25 < 210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 34 agcatcgcat tcagtaccgc ggctg 25 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 35 cgtaagcctc gtcagctatc cgggg 25 <210> 36 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 36 ccaaacgcac cccaacctgt ccgga 25 <210> 37 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 37 gtcgggggta tcgcgttgct ctacg 25 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 38 cggcggtggc attgtcactg ctgct 25 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> Zip code oligonucleotide sequence <400> 39 tcgaattctc ggtgtccgcg ggcga 25 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 40 atgcacccgc ctacggatct gcctc 25 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 41 gacgattggc ggcactggct atcgc 25 <210> 42 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 42 gcagttcgtg gccatggtga ccgct 25 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400 > 43 ccgtcgagtc gggctgctgt tgcaa 25 <210> 44 <211> 25 <212> DNA <213> Arti ficial Sequence <220> <223> Zip code oligonucleotide sequence <400> 44 ttcggagccc gtatcgccgg agtca 25 <210> 45 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 45 tcgggaccga taccaggtag ccggt 25 <210> 46 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 46 agtactggcg tttgcccgct cgctc 25 <210> 47 <211> 25 <212 > DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 47 gagtctgacg ccgcccactt cgact 25 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 48 cgttgtggta gcggcactgg tggtg 25 <210> 49 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 49 tccttgcgtc gcgccgttaa cacgg 25 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 50 gcctcgctgc agtctgcgtt cactc 25 <210> 51 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 51 ctgctgttga gcgtgaagac cgccg 25 <210> 52 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 52 ggtgcttgca gtgctcgggc ccaac 25 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> < 223> Zip code oligonucleotide sequence <400> 53 ctgggtgtgg gtgctcgtac gccga 25 <210> 5 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 54 cgacgcgggc ttggtacgtt tgggg 25 <210> 55 <211> 25 <212> DNA <213> Artificial Sequence < 220> <223> Zip code oligonucleotide sequence <400> 55 gtggtctgcc agccgtcggt gccat 25 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 56 ccggtcgatc gtggtgttcg cggct 25 <210> 57 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 57 catgagcaag ctgcagctgc gcgcg 25 <210> 58 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> Zip code oligonucleotide sequence <400> 58 gttaccgctg gtgctgccgc cggta 25 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 59 acgactgcga ggtgcggtaa gcaca 25 <210> 60 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 60 cggtcgacga gctgccgcgc aagat 25 <210> 61 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 61 gacattcgcg atcgccgccc gcttt 25 <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 62 cggtatcgcg accgcatccc aatct 25 <210> 63 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 63 gctcgaagag gcgctacaga tcctc 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 64 caccgccagc tcggcttcga gttcg 25 <210> 65 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 65 cgactccctg tttgtgatgg accac 25 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 66 cttttcccgt ccgtcatcgc tcaag 25 <210> 67 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 67 ggctgggtct acagatcccc aactt 25 <210> 68 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 68 gaacctttcg cttcac cggc cgatc 25 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 69 tttcggcacg cgcgggatca ccatc 25 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 70 ctcggtggtg ctgacggtgc aatcc 25 <210> 71 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 71 tcaacgtgcc agcgccgtcc tggga 25 <210> 72 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 72 gcgaaggaac tcgacgtgga cgccg 25 <210> 73 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 73 cggggatacc gatctcgggc gcaca 25 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 74 ggagcttacg ccatcacgat gcgat 25 <210> 75 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 75 cgtggcggtg cggagtttcc ccgaa 25 <210> 76 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 76 cgatccaacg cactggccaa accta 25 <210> 77 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 77 ctgaatcctc caaccgggtt gtcga 25 <210> 78 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code olig onucleotide sequence <400> 78 ttcggcgctg gcgtaaagct tttgg 25 <210> 79 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 79 gtaaatctcc agcggaaggg tacgg 25 <210> 80 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 80 ccggctttga actgctcacc gatct 25 <210> 81 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 81 actacgcaac accgaacgga taccc 25 <210> 82 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 82 ggaccaatgg tcccattgac caggt 25 <210> 83 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> cZip code oligonucleotide sequence <400> 83 caacgctgag cgcgtcactg acata 25 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 84 gagacaaagg tctgcgccag cacca 25 <210> 85 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 85 tggccacact gtccatttgc gcggt 25 <210> 86 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 86 ccttgcgacg tgtcaagttg gggtc 25 <210> 87 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 87 aggttagggt cgcgccaaac tctcc 25 <210> 88 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 88 gatgatcgac gagacactct cgcca 25 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 89 gcgatcgccg ggagatatac ccaac 25 <210> 90 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 90 tcgtgccgga ctcgagcacc aatac 25 <210> 91 <211> 25 <212 > DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 91 gctttagcac cgcgatggcg tagac 25 <210> 92 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 92 cagccgcggt actgaatgcg atgct 25 <210> 93 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 93 ccccggatag ctgacgaggc ttacg 25 <210> 94 <211> 25 <212> DNA <213> Artificial Sequence <220> <223 > cZip code oligonucleotide sequence <400> 94 tccggacagg ttggggtgcg tttgg 25 <210> 95 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 95 cgtagagcaa cgcgataccc ccgac 25 <210 > 96 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 96 agcagcagtg acaatgccac cgccg 25 <210> 97 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 97 tcgcccgcgg acaccgagaa ttcga 25 <210> 98 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 98 gaggcagatc cgtaggcggg tgcat 25 <210> 99 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 99 gcgatagcca gtgccgccaa tcgtc 25 <210> 100 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 100 agcggtcacc atggccacga actgc 25 <210> 101 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 101 ttgcaacagc agcccgactc gacgg 25 <210> 102 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 102 tgactccggc gatacgggct ccgaa 25 <210> 103 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 103 accggctacc tggtatcggt cccga 25 <210> 104 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 104 gagcgagcgg gcaaacgcca gtact 25 <210> 105 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400 > 105 agtcgaagtg ggcggcgtca gactc 25 <210> 106 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 106 caccaccagt gccgctacca caacg 25 <210> 107 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 107 ccgtgt taac ggcgcgacgc aagga 25 <210> 108 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 108 gagtgaacgc agactgcagc gaggc 25 <210> 109 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 109 cggcggtctt cacgctcaac agcag 25 <210> 110 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 110 gttgggcccg agcactgcaa gcacc 25 <210> 111 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 111 tcggcgtacg agcacccaca cccag 25 <210> 112 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucl eotide sequence <400> 112 ccccaaacgt accaagcccg cgtcg 25 <210> 113 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 113 atggcaccga cggctggcag accac 25 <210> 114 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 114 agccgcgaac accacgatcg accgg 25 <210> 115 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 115 cgcgcgcagc tgcagcttgc tcatg 25 <210> 116 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> cZip code oligonucleotide sequence <400> 116 taccggcggc agcaccagcg gtaac 25 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR forward primer for codon 72 of p53 <400> 117 atctacagtc ccccttgccg 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR reverse primer for codon72 of p53 <400> 118 gcaactgacc gtgcaagtca 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR forward primer for codon 205 of p53 <400> 119 accatgagcg ctgctcagat 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR reverse primer for codon205 of p53 <400> 120 agttgcaaac cagacctcag 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR forward primer for codon239 of p53 <400> 121 gtgttatctc ctaggttggc 20 <210> 12 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR reverse primer for codon239 of p53 <400> 122 tccaggtcag gagccacttg 20 <210> 123 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Cy5 reporter probe for codon72 of p53 <400> 123 agaggctgct ccccc 15 <210> 124 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Cy3 reporter probe for codon72 of p53 <400> 124 agaggctgct ccccg 15 <210> 125 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Cy5 reporter probe for codon205 of p53 <400> 125 gaaggaaatt tgcgtgtgga gtt 23 <210> 126 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Cy3 reporter probe for codon205 of p53 <400> 126 gaaggaaatt tgcgtgtgga gta 23 <210> 127 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cy5 reporter probe for codon239 of p53 <400> 127 ccatccacta caactacatg tgtg 24 <210> 128 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cy3 reporter probe for codon239 of p53 <400> 128 ccatccacta caactacatg tgta 24 <210> 129 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Capture probe for codon72 of p53 <400> 129 cgtggcccct gcacacgact gcgaggtgcg gtaagcaca 39 <210> 130 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Capture probe for codon205 of p53 <400> 130 tttggatgac agaaacactt ttcgcggtcg acgagctgcc gcgcaagat 49 <210> 131 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Capture probe for codon239 of p53 <400> 131 acagttcctg catgggcgac attcgcgatc gccgcccgct tt 42

Claims (8)

a) amplifying the genes to be tested for SNPs by PCR; b) performing an oligobase linkage analysis method by mixing a reporter probe that combines capture and fluorescent material with the amplified product; c) coupling the product of the above oligobase linkage analysis method to a Zip-Code oligobase chip; And d) SNPs test method comprising the step of reading the SNPs. The method of claim 1, Said Zip-Code oligobase chip is a SNPs test method, characterized in that attached to the Zip-Code oligobase comprising the base sequence described in SEQ ID NO: 1 to SEQ ID NO: 58. The method of claim 1, Said capture source is a method for testing SNPs, characterized in that consisting of the cZip-Code oligobase comprising a nucleotide sequence described in SEQ ID NO: 59 to 116. The method of claim 1, The fluorescent material is Cy2, GFP, YO-PRO-1, YOYO-1, Calcein, FITC, FlourX, ALEXA 488, Rhodamine 110, ABI 5-FAM, Oregon Green 500, Oregon green 488, RiboGreen, Rhodamine Green, Rhodamine 123, Magnesium Green, Calcium Green, TO-PRO-1, TOTO-1, ABI JOE, BODIPY 530/550, Dil, BODIPY TMR, BODIPY 558/568, BODIPY564 / 570, Alexa 546, TRITC, Magnesium Orange, Phycoerythrin R Rhodamine Phalloidin, Calcium Orange, Pyronin Y, Rhodamine B, ABI TAMRA, Rhodamine Red, Cy3.5, ABI ROX, Calcium Crimson, Alexa 594, Texas Red, Nile Red, YO-PRO-3, YOYO-3, R-phycocyanin, C-phycocyanin, TO-PRO-3, TOTO-3, DiD DilC (5), Thiadicarbocyainie, Cy5.5, Cy5 or Cy3 test method characterized in that. The method of claim 1, SNPs inspection method characterized in that the step of reading the SNPs are performed by a chip scanner. a) reagent for PCR; b) reagents for oligobase linkage analysis; And c) SNPs testing kit comprising a Zip-Code oligobase chip for testing SNPs. The method of claim 6, The Zip-Code oligobase chip is a SNPs test kit, characterized in that attached to the Zip-Code oligobase comprising a base sequence described in SEQ ID NO: 1 to SEQ ID NO: 58. The method of claim 6, The reagent for analyzing oligobase linkage SNPs test kit, characterized in that it comprises a capture source consisting of a cZip-Code oligobase comprising a nucleotide sequence described in SEQ ID NO: 59 to 116.