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Probe of human papillomavirus and DNA chip comprising the same

A technology of human papillomavirus and DNA chip, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/testing, etc., and can solve the problem that DNA chips are not yet available for sale

Inactive Publication Date: 2007-09-12
文宇哲
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DNA chips satisfying the above conditions have not been commercially available so far

Method used

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  • Probe of human papillomavirus and DNA chip comprising the same
  • Probe of human papillomavirus and DNA chip comprising the same
  • Probe of human papillomavirus and DNA chip comprising the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1: preparation standard and control specimen

[0109] First, prepare the specimen that will be used as a standard or reference and control in future genotyping checks and analyses.

[0110] The first specimen is a human cervical cancer cell line, which has been confirmed for the presence and type of HPV and has been widely used in the study of HPV genotypes. Human cervical cancer cell lines were purchased from ATCC (Manassas, VA20108, USA) and Korea Cell Line Bank (KCLB) (Seoul University Medical center cancer institute, KOREA), and used after monolayer culture. Specific descriptions of the cell lines are summarized in Table 1.

[0111] The second specimen was from cervical tissue from women who had been diagnosed with cervical cancer or a carcinoma in situ lesion and had undergone surgery. After the specimens were fixed in formalin, the tissues preserved in the paraffin-embedded state were cut into 6-10 microscopic sections with a thickness of 10 μm, stuck...

Embodiment 2

[0114] Example 2: Isolation of DNA

[0115] From the different specimens of embodiment sampling, separate DNA with following suitable method:

[0116] To isolate DNA from cell lines, isolate cell lines cultured in monolayer, put into 50 ml centrifuge tube, centrifuge at 3500 rpm for 30 min, decant supernatant, disperse pellet with 500 μl PBS, transfer to 1.5 ml centrifuge tube, Centrifuge at 12,000 rpm for 2 minutes to remove residual medium. Afterwards, 200 μl of PBS solution and 20 μl of proteinase K (20 μg / μl) were added to the cell pellet and mixed with a vibrating magnetic stirrer. Add 200 μl of GC solution, mix, and solubilize on a 60°C heat block for 20 minutes. After the reaction is complete, add 100 μl of isopropanol to the specimen and mix thoroughly. All solutions were applied to filter cartridges and centrifuged at 10,000 rpm for 1 minute. After discarding the filtrate passing through the column, 500 μl of W1 buffer was added. Then, 500 μl of W2 buffer was add...

Embodiment 3

[0119] Embodiment 3: PCR amplification

[0120] In order to check the genotype of HPV, firstly the E6 / E7 gene and L1 gene of HPV were amplified by PCR, and the human β globin gene was used as an internal control for PCR amplification.

[0121] First, oligonucleotide primers are selected and designed for these PCR amplifications. The primers consist of HPCF / HPCR primers that can detect the E6 / E7 gene of HPV, MY11 and GP6-1 primers (SEQ ID NO.1) that can detect the L1 gene, and human β globulin used to confirm the efficiency of DNA extraction and PCR Gene HBBF / HBBR primer composition. The GP6-1 primer was newly designed, and the other two primers were selected from known primers. The PCR amplification product of the E6 / E7 gene of HPV is about 225 bp in length, the PCR amplification product of the L1 gene of HPV is 182 bp in length, and the PCR amplification product of β globin gene is 182 bp in length. The base sequences of PCR primers for each gene are summarized in Table 2,...

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Abstract

Oligonucleotide probes for analyzing 40 types of HPV were synthesized, and DNA chips were produced by using the oligonucleotide probes. The synthesis of the oligonucleotide probes is based on clones of Ll and E6 / E7 genes of 35 types of HPV obtained from cervical cell specimens from 4,898 Korean adult women and tissue specimens from 68 cervical cancer cases in addition to information based on American and European cases. The DNA chips can analyze the 40 types of HPV found in cervical, diagnose complex infection by at least one type of HPV, and have excellent diagnostic sensitivity and specificity on HPV genetic type up to 100% and reproducibility. Also, the DAN chips are superior to conventional analytic method, and very economical, since they can analyze numerous specimens in shortest time. Accordingly, the DNA chips are useful for predicting cervical cancer and precancerous lesion.

Description

technical field [0001] The present invention relates to probes that bind complementary to the nucleic acid of human papillomavirus (HPV), which is the main cause of cervical cancer and the most prevalent sexually transmitted disease, and to DNA chips or microchips containing the probes. Arrays, diagnostic kits for genotyping HPV, and methods for testing for the presence and genotyping of HPV infection. Background technique [0002] Human papillomavirus (HPV) is a virus with a double-stranded DNA genome surrounded by a capsid, which is shaped like a golf ball. HPV is important to humans in two ways. First, HPV is the most common sexually transmitted disease (STD). It is reported that 50% or more of all adult women will be infected with HPV at least once in their lifetime. Second, HPV infects human epithelial cells, inducing hyperproliferation. In most cases, this hyperplasia is a benign tumor such as simple skin warts, genital warts around the external genitalia or anus, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11
CPCC12Q1/708
Inventor 文宇哲吴明烈任秀彬严泰汉程银惠高丁银裴栽汉
Owner 文宇哲
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