TWI234585B - HURP gene as a molecular marker for bladder cancer - Google Patents

Abstract

Hepatoma up-regulated protein (HURP) gene serves as a useful molecular marker in the detection, preliminary screening and monitoring of bladder cancer in a human subject. A method for the detection of bladder cancer, in particular transitional cell carcinoma (TCC) of the bladder, in a human subject is disclosed, in which the detected expression of the human HURP gene in a sample taken from a subject suspected to have bladder cancer is indicative of the presence of the bladder cancer. The present invention further provides a non-invasive method for the detection, preliminary screening or monitoring of urinary TCC in a suspected subject, in which a urine sample taken from the subject is analyzed to determine an expression of the human HURP gene, the presence of which is indicative of the presence of urinary TCC.

Description

1234585 发明 Description of the invention (the description of the invention should be stated ... the technical field to which the invention belongs, the prior art, the content, the embodiments and the drawings are briefly explained) [Technical Field to which the invention belongs 3 Field of the invention The present invention relates to a human liver cancer rise -Regulated protein 5 (HURP) gene is used as a molecular marker in the detection, preliminary screening and monitoring of bladder cancer in a human individual, of which a sample was obtained from an individual suspected of having bladder cancer The expression of human HURP gene detected in it is a sign of the presence of bladder cancer. The present invention therefore provides an effective method for detecting bladder cancer in a human individual, especially bladder metastatic epithelial cancer (TCC). The invention further provides a non-invasive method with high accuracy and convenience for detecting, preliminary screening and monitoring udnary TCC in a suspected individual, one of which is taken from a suspected human Individual urine samples were analyzed to detect the expression of the human HURP gene, and the presence of this gene expression was characterized by the presence of urinary TCC. Description of related skills: Urothelial carcinoma is the second most common malignant tumor of the urinary tract, and it ranks second among all urinary tract tumors. Dead 20 waves [Konety BR and Getzenberg RH: Urine based markers of urological malignancy. J Urol 165: 600-611, 2001) 〇 Commercially defeated metastatic epithelial cancer (TCC) is associated with more than 90% of urinary epithelial neoplasms, and it represents mastoid surface damage with low malignant potential Or it can be highly aggressive and lethal. When bladder cancer was detected in a localized stage by stage I 1234585 发明, invention description, the 5-year survival rate was 94%. Once the disease has spread regionally or remotely, the five-year survival rate will drop to 49% and 6%, respectively. {Droller MJ: Individualizing the approach to invasive bladder cancer. Contemp. Urol, July / August, pp, 54-61, 5 7990). Therefore, it is important to detect the early signs of surface cancer recurrence before cancer cells have time to change their behavior and become invasive. Traditional cytology and cytoscopy under a scheduled follow-up procedure is the primary method for surveillance of patients with urinary τ c C. The low sensitivity of cytology to the use of excreted urine for low-level tumors requires regular use of 10 to non-invasive cytoscopy, thus causing associated discomfort caused by urethral instruments during cytoscopy And the potential danger of infection. The development of a sensitive non-bismuth aggressiveness test that specifically detects bladder cancer at an early stage will improve clinical results by starting treatment earlier.

15 The invention name is "Methods and Probe Sets for Detecting Cancer", US 6,376,188, the invention name is "Methods for Detecting Nucleic Acid Sequences in Urine", 0,251,638 and the invention name is "A method for protecting nucleic acid sequences in urine, US 6,287,820 has separately disclosed a bladder cancer test using a set of chromosomal probes to detect a urine sample. 10 The name of the invention is` `Comparison Sexual somatic heterozygosity (CGH), US 6,335,167 discloses a bladder cancer test by detecting the number of copies of a nucleic acid sequence, specifically detecting a unique sequence in a test sample in at least one selected from the following groups Amplification of positions. Q2 i of human chromosome 8; q31-qter of human chromosome 3; pl5__ of human chromosome 7; human chromosome 8 of 1234585 玖; q24-qter of the invention description; cenpi3 of human chromosome n As well as the ql3-qter of human chromosome 9. The US patent 6,291,163 disclosed in the invention titled "Methods for Detecting Cell Proliferative Disorders," examines 5 of 5 in a body by detecting nucleic acid sequences. Cancer (including bladder cancer) and precancerous cancer are characterized by the following steps: 扩增 Amplify the test sample DNA at a gene position that is a heterogeneous embryo for the individual, where the gene position contains the Two dual genes, the gene position contains a microsatellite surface, wherein the test sample surface is from a cell flowing into an organ in the test sample, wherein the test 10 test sample is selected from the individual and includes the following group Group: urine, sputum, bile, feces, saliva, tears, serum and plasma; and 0) By comparing the level of microsatellite DNA present at the first dual gene with respect to the position at the second dual gene The level of the microsatellite leg is used to detect a dual gene imbalance at the gene position, and one of the dual gene imbalances is characterized by cancer or precancerous. The invention titled "Method and Device for Early Detection of Bladder Cancer in Urine Samples WO 01/86288 discloses a method for early detection of bladder cancer in a urine sample, which comprises extracting from The step of amplifying the RNA of the cells in the urine. This is achieved by using a marker of the message RNA for the catalytic component of 20 enzymes at the chromosome end and a & actin to prove the easy access of the RNA. And as a criterion for quantitative assessment, combined with at least one additional marker selected from the group: a marker for a protein belonging to the cytokeratin family, and a suitable for detecting inflammation associated with neoplastic infiltration Lymphocyte labeling of cells; and a final step to detect a substance that has been expanded to 1234585% and described in the invention. WO 99/63110, titled "Testing and Treatment of Cancer", discloses a method for testing a human patient. A method of bladder cancer, comprising the steps of: obtaining a sample containing nucleic acids and / or proteins from a patient's bladder, preferably from urothelium 5; And (ii) testing whether the sample contains a Pax 5 nucleic acid or protein associated with bladder cancer. WO 02/27329, which was invented as "Biomarker for Bladder Metastatic Epithelial Cancer", can be used as bladder metastasis An epithelial cancer (TCC) test, one of the auxiliary adjuvant protein markers, methods and sets. The case uses markers that differ 10 times in samples from TCC patients and a control group (such as individuals with no TCC). US 6,335,170 discloses a method for detecting the expression of a urethral epithelial or bladder cancer cell, comprising: detecting the expression of one or more genes in a sample containing a urethral epithelial or bladder cancer cell, whereby, a The performance of the fifteenth aspect is formed; the performance of the first aspect is deducted from the performance of a second aspect, wherein the second aspect is mainly by using the one or more genes and a Mucosa, smooth muscle, or connective tissue cells are formed from a sample. The subtraction step forms a third aspect that reflects the performance of urethral epithelium or bladder cancer cells, and is present in the sample. The proportion of subcutaneous mucosa, smooth muscle, or connective tissue cells is not involved. Other related studies on bladder cancer include: Konett BR and Getzenberg RH: Urine based markers of urological malignancy. J Urol 165: 600-611, 2001; Droller MJ: Individualizing the approach to invasive bladder cancer. Contemp. Urol., 1234585 玖, invention description

July / August, pp. 54-61, 1990; Nomura N, Miyajima N, Sazuka T, et al: Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA 5 clones from human immature myeloid cell line KG-1. DNA Res 1: 27-35, 1994; Bassal S, Nomura N, Venter D, et al:

Characterization of a novel human cell-cycle-regulated homologue of Drosophila dlgl. Genomics 77: 5-7, 2001; Ellis WJ, Blumenstein BA, Ishak LM, et al: Clinical evaluation of the 10 BTA TRAK assay and comparison to voided urine cytology and the Bard BTA test in patients with recurrent bladder tumors. The Multi Center Study Group. Urology 50: 882-887, 1997; Sarosdy MF? Hudson MA, Ellis WJ, et al: Improved detection of recurrent bladder cancer using the Bard BTA stat Test. Urology 50: 349-15 353, 1997; Seripa D, Parrella P, Gallucci M? Et al: Sensitive detection of transitional cell carcinoma of the bladder by microsatellite analysis of cells exfoliated in urine. Int J Cancer 95: 364-369 , 2001; Ponsky LE, Sharma S, Pandrangi L, et al: Screening and monitoring for bladder cancer: refining the use of 20 NMP22. J Urol 166: 75-78, 2001; Konety BR, Nguyen TS, Dhir R, et al : Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4. Clin Cancer Res 6: 2618-2625, 2000; Rote m D, Cassel A, Lindenfeld N, et al: Urinary cytokeratin 20 as a marker for transitional cell carcinoma. Eur Urol 37: 601-604, 2000; 10 1234585 发明, description of the invention

Sanchez-Carbayo M? Urrutia M, Gonzalez de Buitrago JM? Et al: Evaluation of two new urinary tumor markers: bladder tumor fibronectin and cytokeratin 18 for the diagnosis of bladder cancer. Clin Cancer Res. 6: 3585-3594, 2000; Sanchez -Carbayo 5 M, Urrutia M, Silva JM, et al: Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer. Urology 55: 526-532, 2000; Smith SD? Wheeler MA, Plescia J, et al: Urine detection of survivin and diagnosis of bladder cancer. JAMA 285: 324-328, 2001. 10 In any case, to the best of the applicant's knowledge, none of the literature cited above has reported the expression of a HURP gene in bladder cancer or urinary epithelial cancer. Applicants have surprisingly found that TCC tissue samples exhibit reproducible and significant performance of HURP mRNA transcripts. Based on this discovery, it is possible to develop a human HURP gene for detection, preliminary screening, or analysis by analyzing the performance of a human HURP gene in a biological sample (such as 15 tumor tissue and excreted urine). An accurate, convenient and non-invasive method for monitoring bladder cancer in a human individual. SUMMARY OF THE INVENTION 3 Summary of the Invention 2 Therefore, in a first aspect of the present invention, the present invention provides a method for detecting bladder cancer in a human individual, comprising the following steps: From an individual suspected of having bladder cancer Obtaining a biological sample; and detecting a human liver cancer ascending-regulated protein (HURP) gene in the 1234585 玖, description of the manifestation of the sample, the detected manifestation of the human HURP gene is the presence of bladder cancer Characterization. In a second aspect, the present invention provides a method for monitoring bladder cancer in a human individual, comprising the following steps: 5 obtaining a biological sample from an individual suspected of having bladder cancer in a period of time; and, The expression of a human HURP gene in the sample was measured. The detected expression of the human HURP gene was a sign of the presence of bladder cancer. In a second aspect, the present invention provides a primer set for detecting bladder cancer in a human individual, which comprises a sequence selected from a sequence identification number: 2 and a sequence identification number: 4 A forward primer of the nucleotide sequence of the DNA sequence, and a reverse primer having a nucleotide sequence selected from the sequence identified by the sequence identification number · 3 and the sequence identification number: 5. In a fourth aspect, the present invention provides a test kit for detecting bladder cancer in a human individual 15, comprising at least one primer pair having a forward primer and a reverse primer, each primer having a Nucleic acid sequence t · Heterozygous to one with one nucleus; the acid sequence corresponds to at least three nucleotides of the human HURP gene of sequence identification number: -· The above and other purposes, features, and advantages of this ignorance will become apparent after referring to the detailed description and preferred embodiments of the following ~ 20 and the drawings attached with the text, where: The drawings briefly explain the first figure Shows the full length of the nucleotide sequence of the human HURp gene (sequence recognition editor: 1) 'wherein it is complementary to 12 of the 12 primers designed according to the present invention 1212585 玖, the description of the invention is underlined and bold Figure 2 shows a restriction map of the vector pET32a (Novagen Inc.) used in the following examples, in which a restriction site of 5 am//I was used to insert a full-length nucleotide of the human HURP gene Sequences; Figures 3A and 3B show HURP and cold-actin reverse transcriptase polymerase chain reaction (RT-PCR) analysis in TCC tissues, respectively, where N = tumor adjacent tissue, T = tumor tissue , And 294, 306, 307, 315, 317, 319, 320, 321, 322, 327, 335, and 337 = patient numbers; 10 Figure 4 shows 5 photos taken from patients with benign prostatic hypertrophy Human HURP Gene Table in Gland Urinary Epithelial Samples RT-PCR analysis of β-actin (307 bp) in the left half and HURP (370 bp) in the right half; Nl, N2, N3, N4, and N5 = 5 benign prostatic hypertrophy (BPH) samples; NC = negative control group; and M represents a molecular weight marker of base pair 15; and Figures 5A and 5B show HURP and β-muscle in void urine samples, respectively. RT-PCR analysis of kinesin, in which 2, 6, 7, 8, 14, 15, 20, 5, 10, 11, 12, 13, 16 and 18 = patient number; NC = negative control group; and M represents Molecular weight markers in base pairs. [Embodiment] Detailed description of the invention Genes and proteins involved in cell cycle regulation and apoptosis have been found to be important in the development of cancer. For the confirmation of the components of the cells that will control the cell cycle and apoptosis, there is a need in the art for the need for continuity. Human hepatocellular carcinoma ascending-regulated protein f (HURp) gene _Βι deposited 5 # 10 side 7 secrets with the full-length nucleotide sequence as described in the sequence identification number: 1 Zhou Zhou and others waited for liver cancer The research identified a novel cancer-related gene involved in cell growth. In Zhou's successful research on hepatocellular carcinoma, there is a study on novel genes, particularly in human cDNA banks, and cell cycle-dependent performance in microarray database. Cell cycle regulatory genes (c Tao was identified as a hepatocellular carcinoma ascending-regulated protein (HURp) gene ⑽⑽ et al., The manuscript was submitted for publication). It ’s amazing if you have discovered for the first time that the human HURp gene can be used as a powerful molecular marker for bladder cancer, and it can use the human HURP group @ 用 —money-invasive bladder test to detect sensitively low Degree of bladder tumors and specifically exclude most non-cancer 15 or clinically non-significant patients. According to the present invention, here is provided a bladder cancer for detecting, preliminary screening or monitoring in a human individual, comprising the following steps: obtaining a biological sample from an individual suspected of having bladder cancer; and detecting a The expression of a human liver cancer up-regulated protein (HlJRP) gene in this sample. The detected expression of the human HURP gene is a sign of the presence of bladder cancer. The method in this case can be applied to the detection, preliminary screening or monitoring of transitional epithelial cancer (TCC) of the bladder. 14 1234585 (ii) Description of the invention Preferably, the method of the present invention is performed using a biological sample containing cells selected from the group consisting of urinary epithelial cancer cells, bladder cancer cells, or a combination of the two. Preferably, the biological sample is selected from the group consisting of 5: urine, urinary epithelial biological specimen, gold fluid, plasma and serum. In a preferred embodiment of the present invention, the biological sample is urine.

The human HURP gene has a nucleotide sequence as described in FIG. 1 and the sequence identification number: 1, and according to the nucleotide sequence, it can be hybridized to at least 13 nucleotides of a human HURP gene. Nucleotides can be designed to be used as a test probe or primer to detect the performance of the gene using traditional methodologies well known in the biotechnology arts. As an example, referring to FIG. 1, the applicant has designed four primers that can be used in the method for detecting bladder cancer in this case, which include: the first primer pair: 15 forward primer l- > 5 ' -ggatccaatagacactttggtttg-3 '(sequence identification number: 2)

Reverse primer l-^ '-ggatcccacctttccttctggttd (sequence identification number: 3) second primer pair: forward primer 2-5'-caacgaaaacagatgctc-3' (sequence identification number: 4) reverse primer 2- > 5'- tgagtagctgatcgagtc-3 '(sequence identification number: 5) 20 It is expected that the forward primer 1 can be paired with the reverse primer 2, and the forward primer 2 can be paired with the reverse primer 1. According to the present invention, the expression of the human HURP gene is detected by analysis of an mRNA transcript from the human HURP gene or an analysis of the protein translated from the human HURP gene t 15 1234585 (invention description mRNA transcript). Detection of proteins translated from the mRNA transcripts of the human HURP gene can be performed using traditional methodologies well known in the biotechnology arts. 5 In a preferred embodiment of the present invention, the detection of human HURP gene expression is performed by detecting the presence of the human HURP gene mRNA transcript in the biological sample.

Preferably, the presence of the mRNA transcript of the human HURP gene is performed using at least one of the following methodologies: hybridization, cycling probe reaction (PCR), polymerization peak chain reaction (PCR), Nested PCR, reverse transcriptase polymerase chain reaction (RT-PCR), multiplex PCR polymerase chain reaction-single-stranded structure polymorphism, ligase chain reaction (LCR), mainly limiting fragment length polymorphic nucleic acid sequences Amplification reaction (NASBA), and transcription-regulated amplification reaction (TMA). 15 In a preferred embodiment of the present invention,

Detection of the presence of mRNA transcripts was performed using RT-PCR. In a preferred embodiment of the present invention, the detection of the presence of the mRNA transcript of the human HURP gene is performed by the following steps: (a) total RNA is extracted from the biological sample; 20 (b) will be derived from The extracted RNA of step (a) is introduced into a reverse transcription polymerase chain reaction (RT-PCR) process using at least one primer pair having a forward primer and a reverse primer, each primer having a nucleotide sequence Will hybridize to a human HURP gene with a nucleotide sequence corresponding to sequence identification number: 1 at least 16 1234585 585, invention description 13 nuclei; acid; and (c) detect if there is a nucleotide sequence that is identical The RT-PCR product that is part of or complementary to the nucleotide sequence of the human HURP gene has been generated from the RT-PCr treatment of step (1 ^). The presence of the 5 RT-PCR product is a sign of the presence of bladder cancer. Preferably, each primer used in step (b) will hybridize to at least 8 nucleotides of the human HURP gene. Preferably, the at least one primer pair used in step (b) Contains an optional sequence identification number : 2 and sequence identification number: the forward primer of the 10-sequence nucleotide sequence described in 4, and a nucleotide sequence having a sequence selected from the sequence identification number: 3 and the sequence identification number: 5 Preferably, the at least one primer pair used in step (b) has a forward primer corresponding to a nucleotide sequence corresponding to the sequence identification number: 4 and

15 A reverse primer with-corresponding to the sequence identification number: 5 _Osac acid sequence. In the present invention-the preferred implementation, the RT-PCR processing package in step 3 3. A first using a first primer pair An amplification reaction, the -primer pair having a first forward primer having a nucleotide sequence corresponding to the sequence identification number and a first reverse primer having a nucleotide sequence corresponding to the sequence identification number A forward primer; and a second amplification reaction using a second primer pair, the second primer pair having _ two second forward primers with a nucleotide sequence corresponding to the sequence identification number: 4 and a 17 20 1234585 发明 Description of the invention The reverse primer has a second nuclear sequence corresponding to the sequence identification number: 5 According to the present invention, a primer is provided here, a primer for bladder cancer in a human individual Group, which contains eight selectable sequences from the sequence identification number • Γ sequence identification number: the forward primer of the sequence of ㈣ 醆 of the sequence described in ^: has-selected from the sequence identification number: 3 and the sequence identification number: 5 acid sequence Reverse primer. In a preferred embodiment of the present invention, the forward arch element has a gallic acid sequence as described in the sequence identification number: 2. 10 15 In a preferred embodiment of the present invention, the forward primer has a gallic acid sequence as described in the sequence identification number: 4. In a preferred embodiment of the present invention, the reverse primer has a nucleotide sequence as described in SEQ ID NO: 4. In a preferred embodiment of the present invention, the reverse primer has a nucleotide sequence as described in the sequence identification number_ · 5. According to the present invention, a test kit for detecting bladder cancer in a human individual is provided herein, which comprises at least one primer pair having a forward primer and a -reverse primer. Each primer has a nucleus? The acid sequence will be hybridized to at least 13 nucleotides of the human HURP gene having a nucleotide sequence corresponding to the sequence identification number: i. In another preferred embodiment of the present invention, the test set includes a first forward primer having a nucleotide sequence corresponding to a sequence identification number: 2 and a first forward primer having a sequence identification number: 3 No. 18 20 1234585 of the nucleotide sequence, invention description, a first primer pair of a reverse primer, and a second forward primer having a nucleotide sequence corresponding to the sequence identification number 5 Tiger: 4 and a ^ A pair of deer second primers of the second reverse primer of the nucleotide sequence of sequence identification number: 5. 5 In addition to the above, those skilled in the art will understand that various applications falling within the scope of the present invention include the use of any form of evaluation analysis. For example, RNA and proteins can be isolated and analyzed from a test sample using techniques known in the art. For example, they can be separated from fresh or frozen biological specimens, tissues fixed in formalin, and body fluids such as blood, plasma, serum, urine, or saliva. Although RT-PCR can be used, other techniques are also expected. These include other techniques for the analysis of specific mRNA species, including PCR, nested PCR, in situ hybridization, northern dot blotting, high-density performance arrays, microarray 15 columns, and techniques for analysis of specific protein products. , Such as enzyme-linked immunosorbent assay (ELISA), Western blotting and enzyme analysis. The present invention will be described in more detail with reference to the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the present invention. 20 Example i: RT_PCR analysis of hurp gene expression Experimental materials and methods Patient characteristics 80 TCC samples taken from the urethra were obtained from March 1998 to 19 1234585 发明 Description of invention at the Chi Mei Medical Center in September 2001 Obtained by cystectomy and urethral tumor resection. Distal overall normal tissue was also obtained for analysis and these were treated as tumor-adjacent tissue samples. All tissue samples were frozen in liquid nitrogen and stored at -86 ° C for different periods of time 5. A representative portion of each frozen block was embedded in paraffin and stained with hematoxylin-eosin, and reviewed by a pathologist to assess the tumor stage in the sample. All specimens were graded using one of the World Health Organization amendments, and the pathology stage was based on a TNM pathology stage system such as > 7 //, 10 MB, Reuter VRf et al: The World Health Organization / International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol 22: 1435-1448, 1998; and 15 Chisholm GD, Hindmarsh JR, Howatson AG, et al: TNM (1978) in bladder Cancer: use and abuse. Br J Urol 52: 500-505, 1980) The stage of the bladder tumor shows how deep the tumor has invaded. Surface tumors are called Ta, and T1-4 is used to describe the 20 degree elevation that penetrates into the muscle. The grade of a bladder tumor is expressed on a scale of I-IV (1-4). This grading reflects the cytological characterization of the cells. Among them, cells of level I are almost normal, cells of level II are somewhat abnormal, cells of level III are obviously abnormal, and cells of level IV are highly non-normal. normal. 20 1234585 发明, Description of the invention RNA isolation Total RNA is isolated from frozen tissue specimens or urine sediment using the Ultraspec ™ RNA isolation system (Biotecx Laboratories Inc., Houston, Texas). In a hand-held glass-Teflon tube 5, approximately 0.5-1.0 g of tissue is homogenized in 1 ml of Ultraspec ™ reagent. After homogenization, the homogenate was stored at 4 ° C for 5 minutes, then extracted with 0.2 ml of chloroform, and then centrifuged at 12,000 g (4 ° C) for 15 minutes. The RNA in the aqueous layer was precipitated with an equal volume of isopropanol and washed twice with 75% alcohol. The content of RNA10 was estimated by spectrophotometry (1 RNA with an OD value equal to 40 # g / ml measured under A260), and the purity of the extract was estimated using the ratio of A260 / 280, and In all cases it is greater than 1.75. To collect urine, 50 ml of clean urine was obtained on the patient's first excretion of the day. The samples were centrifuged at 3,000 g (4 ° C) for 20 minutes. The fine cell sinks were mixed with 1 ml of Ultraspec ™ reagent and extracted using the same method as above. Reverse transcriptase-polymeric coupling reaction

Single-stranded complementary DNA (cDNA) was treated at 42 ° C with 1 μΐ Superscript II reverse transcriptase (Life Technologies Inc., 20 Gaithersburg, MD) with ligor-dT primary contacting (1 pg). The total RNA lasted 50 minutes. After heating at 70 ° C for 15 minutes, a first amplification reaction was performed using Taq polymerase buffer containing the following: 1/10 volume of reverse transcribed RNA, 200 μπιοΙ / L 21 1234585 发明, description of the invention dNTPs, 1 U DyNAzyme ™ II DNA polymerase, and 10 fl Μ of each human HURP gene primer [5'-GGATCCAATAGACACTTTGGTTTG-3 '(forward) and 5'-GGATCCCACCTTTCCTTCTGGTTC-3, (reverse)], and at 94. Reaction denaturation at 5 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extended reaction at 72 ° C for 1 minute, 30 cycles, followed by incubation at 72 ° C for 5 minutes . For nested PCR of urine specimens, 1/10 of the first amplified product was directed to a nested HURP primer 5, -CAACGAAAACAGATGCTC-10 3 '(forward) and 5'-TGAGTAGCTGATCGAGTC-3' (reverse ) The second round of amplification reaction, and the denaturation reaction at 94 ° C for 1 minute, the annealing at 60 ° C for 1 minute, and the extended reaction at 72 ° C for 1 minute, 30 times Cycle and incubate at 72 ° C for 5 minutes. 15 The routine RT-PCR control group without reverse transcriptase (RT) as a template was used as a negative control group, and by inserting a full-length HURP nucleotide sequence into the pET32a (+) vector (Novagen Inc.) and pET32a-HURP was constructed as shown in Figure 2 as a template is a positive control group. The amplified product was separated with 0.1 tons of GeneRuler ™ 100bp DNA ladder 20 label (MBI Fermentas, Lithuania) on a 1.5% agarose gel using electrophoresis, and stained with ethidium bromide for visual observation. Data quantification and standardization To quantify the mRNA expression level of the HURP gene, all films are based on 1234585 玖, invention description

Image Master ™ VDS Video Imaging System (Amersham Pharmacia

Biotech Inc., Piscataway), and recorded using LISCAP image capture software (Amersham Pharmacia Biotech Inc_, version 1.0). The optical pixels of the PCR products were quantified to arbitrary units using ImageMaster ™ TotalLab 5 software (Amersham Pharmacia Biotech Inc., version 1.11). To compare the samples tested, standardization of the data is necessary. For this purpose, the PCR amplification product of β-actin was used as an internal standard to represent a fairly equivalent number of cDNA templates that were introduced into the PCR. The mean value is expressed as the value standard deviation and compared using Student's test. Results: Representative representative patterns of HURP genes in TCC tissue samples and control individuals are shown in Figure 3A. In 11 groups (patient numbers: 294N / T 15, 306N / T, 307N / T, 315 N / T, 317 N / T, 319 N / T, 320 N / T, 321 N / T, 327 N / T (335 N / T, 337 N / T), considerable mRNA transcripts were amplified in the tumor portion (see Figure 3A). Only one group of tissue samples (patient number: 322 N / T) showed no detectable transcript of the HURP gene in both cases. As an internal standard, the PCR amplification product of β-actin 20 protein (see Figure 3B) was used to represent a fairly equal number of cDNA templates that were introduced into the PCR. Example 2: Evaluation of tumor specificity of HURP gene expression by RT-PCR analysis 23 1234585 发明, invention description To detect whether the overexpression of HURP is tumor specific, from benign prostate hypertrophy (BPH) patients HURP transcripts of the prostate urethral epithelium were amplified using RT-PCR. These experiments were performed using the procedure as described in Example 1 above, except that HURP transcripts from prostate urethral epithelial cells from benign prostatic hypertrophy patients were expanded using RT-PCR. Increase to detect whether the overexpression of HURP is tumor-specific.

Fifteen specimens from the prostate urethral epithelium of BPH patients with normal cytoscopy were collected and referred to RT-PCR. Referring to Figure 4, β-actin 10 transcripts were equally amplified from 5 representative samples of BPΗurethral epithelium, but no HURP transcript was detected in these samples. In addition to these BPH samples, a non-bladder control group (containing 4 samples from the liver and 4 samples from the heart) was tested and no HURP transcripts were detected in these tissues (data not shown).

15 Example 3: Evaluation of tumor stage by RT-PCR analysis of HURP gene expression To detect a possible relationship between HURP gene overexpression and tumor progression, 45 pairs of tissue samples containing a TCC and a counterpart of a tumor adjacent tissue Human HURP transcripts were analyzed and standardized with β-actin transfection 20 records. Referring to Table 1, it is noted that the amount of HURP genes expressed in 35 tumor adjacent parts (35/45, 77.8%) is lower than that of the tumor part (group I, ρ < 0 · 0001), and there are 10 tumors adjacent Some (10/45, 22.2%) showed 24 1234585. The invention description showed that the HURP gene (group II) was almost identical to the tumor part. Eighty TCC patients whose pathology was identified as 0 series I, 37 series π, 31 series III, and 12 series IV were collected, and the human HURP expression ratio in the tissue samples was collected. They are listed in Table 2. It appears that there is no significant correlation between 5 HURP performance and tumor progression. Of the 9 HURP-negative tumors, 6 were grade II, 1 was grade mountain, and 2 was grade IV.

Table 1 HURP performance ratio of TCC tumors and adjacent tissues HURP performance ratio-group number (standardized by β-actin) Mean ± standard deviation TCC tumor tissue tumor adjacent tissue I 35 0.658 + 0.061 0 · 070 ± 0.085卞 Π * 10 0.820+ 0.275 0.770+ 0.327: HURP relative to β · actin (multiple of optical pixels) ρ < 0.0001 10

HURP performance ratio for TCC patients HURP-positive (%) HURP-negative (%) HURP performance values — (standardized by β-actin) Mean ± standard deviation TCC, grade I 0 — — — TCC, grade Π 37 3 1/37 (83,8) 6/37 (16.2) 0_56 ± 0.394 TCC, grade plate 31 30/3 1 (96.8) 1/31 (3.2) 0.59 ± 0.309 TCC, grade Number IV 12 10/12 (83.3) 2/10 (16.7) 0.54 ± 0.129 Total 80 71/80 (88.8) 9/80 (11.2) 0.55 ± 0.369 25 1234585 发明 Description of the invention Example 4: HURP gene expression as Evaluation of one of the biomarkers for TCC testing To evaluate the potential use of urine-HURP as a novel molecular marker for urine for the detection of urinary TCC, 7 additional patients with 5 TCC with newborn or relapsed by using RT-PCR was used to analyze urine-HURP. The total RNA was extracted from the urine cell pellet and reverse transcribed by initial contact with Oigo_dT. Amplification reaction was specifically induced by HURP

Or β-actin specific primers. A 37-base-pair cDNA was amplified from the urine cell pellet of all patients with TCC10 (Figure 5A). In contrast, urine cell pellets from 7 additional patients (3 with urinary tract infection, 2 with chronic urethral bladder inflammation, 1 with renal cell carcinoma, and 1 with BPH) did not have HURp transcripts. In a controlled experiment, a 307 base pair of β-actin < DNA fragment consists of

Urine in the control group and patients with TCC was indistinguishably enlarged (Figure 5B). When all the patents and documents cited in this book are incorporated into the case as a whole as a reference 1, the description (including the definition) in this case will prevail. For the description, it is obvious that many modifications can be made and the patent application attached to the document, although the present invention has been changed with reference to the above specifics without departing from the scope and spirit of the present invention. It is therefore intended that the present invention be limited only by those shown in the scope. 26 20 1234585 发明 Description of the invention Sequence Listing < 110 > Chi Mei Foundation Medical Center Yangming University Department of Life Sciences Zhou Chenggong 5 Qiu Wenxiang Huang Yulun < 120 > HURP gene as a molecular marker for bladder cancer < 13〇 > NP-17020 -TWN 10 < 160 > 5 < 170 > Patentln version 3.1 15 < 21〇> 1 < 211 > 2 97 9 < 212 > DNA < 213 > (Homo sapiens) 20 < 4 00 > 1 agcaaaccaa tcgcaagcct cgt tgagtgg aaggggtggg atc 11ccccg gaagt 11 tgg 60 11 aaagcccc t ccaa t cage ggctcggtgc ggcaagt t tg aat t tcgtgg aggctcgggt no 25 tgtgagggt t cctgct tegg agtcggcggt ggtcgtccag accgagtgt t ctttactttt 180 tgt t tggt tg aggt t teaeg ctagaaggtg gctcaggatg tet tcatcac a 111 tgccag 240 30 tcgacacagg aaggatataa gtactgaaat gat tagaact aaaat tgc tc ataggaaatc 300 actgtctcag aaagaaaata gacataagga ataegaaega aa t agacac tt tggt tggaga taga 11 tga aca tga aga 11a ta t tctaggtga 480 27 1234585 发明, description of the invention t caacgaaaa caga t gc t cc aaaaa t acaa agaagaaaag caac 11 caaa aa 11gaaaga 540 5 gcagagagag aaagctaaac gaggaatat t taaagt cga 11 tgt gt cgt 660 t tctgtacgg a 11acaaggt caaaggccaa agaccaaa tg gageagae ta agat tgataa 720 10 cgagagtgat g 11 egageaa t ccgacc tgg t ccaagacaa ac 11 c tgaaa agaaagtgtc 780 agacaaagag aaaaaagt tg tgcagcctgt aatgcccacg tegt tgagaa tgactcgatc 840 age tac t caa gcagcaaagc aggt tcccag aacag tct ca tct accacag caagaaagcc 900 15 ag t cacaaga gctgctaatg aaaacgaacc agaaggaaag gtgccaagta aaggaagacc 960 tgccaaaaat gtagaaacaa aacccgacaa gggtat t tet tgtaaagtcg atagtgaaga 1020 20 aaa t ac 111 g aa 11 cacaaa ctaatgcaac aagtggaatg aatccagatg gagtet tatc 1080 aaaaa t ggaa aac 11 acc tg aga t aaa t ac tgcaaaaa ta aaagggaaga a 11 cc 11 ege 1140 acctaaggat 11 tatgt t tc agccac tgga tggtctgaag acctatcaag taacacct at 1200 25 gac t cccaga ag t gccaa tgc 111111 gac acccag11 ac acctggactc ct t taaaaac 1260 agaagt tgat gag tct caag caacaaaaga aa 1111ggea caaaaa tgta aaac 11 ac tc 1320 30 t accaagaca at acagcagt t11 aacg t tggcatgaa gaacatgt 11 taaataaaaa tgaage t ac t ac t aaaaa 11 taaatggcct 1440 t ccaa t aaaa gaag t ccca t cac t tgaaag aaatgaaggt egaat tgc tca aca 11 gt ag 1560

281234585 Jiu, the invention described ct tcgagtgg gacaggaaac t tgaat tgga cat tccagat gatgetaaag atet tat teg 1620 5 cacagcag11 gg t caaacaa gactccttat gaaggaaagg 11 taaacagt ttgaaggact 1680 ggt tgatgat tgtgaa ta ta aacgaggtat aaaggagac t acctgtacag atctggatgg 1740 at 11 tgggat atggt tagt tt tcagataga agatgtaatc cacaaat tea acaatctgat 1800 10 caaac 11 gag gaatctgggt ggcaag t caa t aa t aa tatg aatcataata tgaacaaaaa 1860 tgtct t tagg aaaaaagt tg tc tcaggtat agcaagtaaa ccaaaacagg atgatgctgg 1920 aagaat tgca gcgagaaatc gcctagctgc ca taaaaaa t gcaatgagag agagaat tag 1980 15 gcaggaagaa tgtgctgaaa cagcagt t tc tgtgatacca aaggaagt tg ataaaatagt 2040 gt tcgatgct ggat 111 tea gagt tgaaag tcctgt taaa ttattctcag gactttctgt 2100 20 ctct tctgaa ggcccttctc aaagac t tgg aacacc taag tctgtcaaca aagctgiatc 2160 t cagag t aga aatgagatgg gcat tccaca acaaac taca tcaccagaaa atgccggtcc 2220 tcagaatacg aaaagtgaac atgtgaagaa gac 11 tgt 11 t tgagtat tc ctgaaagcag 2280 25 gagcagcata gaagatgctc agtgtcctgg at taccagat t taat tgaag aaaaccatgt 2340 tgtaaataag acagac t tga aggtggat tg 11 tatccagt gagagaatga gt t tgcctct 2400 30 tct tgctggt ggagtagcag atgatat taa tac taacaaa aaagaaggaa 11 tcagatgt 2460 tgtggaagga atggaactga at tct tcaat tacatcacag gatgt 11 tga tgagtagccc 2520 tgaaaaaaat acagc t tcac aaaa t agea tct tagaagaa ggggaaacta aaat t tc tea 2580 35 gtcagaacta 11 tgataata aaagtc tcac tac tgaatgc cacct tct tg at tcaccagg 2640

29 1234585 发明, description of invention tctaaactgc agtaatccat 11 ac t cage t ggagaggaga ca t caagaac at gccagaca 2700 5 cat t tct 111 ggtggtaacc tgat tact 11 t tcacctcta caaccaggag aat 11 tgaat 2760 t taaaaataa atccaaac taga tat tat ta 2820 t tgaaa 1111 ggagaaaa tg tat t tgtgt t cac 11 c ta ta gcatataatg 111 taatat t 2880 10 ctgtgt teat caaagtgtat 11 tagatata ctct t tetea agggaagtgg ggatat 11 tg 2940 tacat 11 tea acacacata 2 agt agt ; 211 > 23 < 212 > DNA < 213 > artificial sequence < 220 > 20 < 223 > HURP amplification < 4〇〇 > 2 ggatccaata acact t tggt ttg 23 25 < 21〇 > 3 < 211 > 23 < 212 > DNA 30 < 213 > artificial sequence < 220 > < 223> HURP amplification < 4〇〇 > 3 35 cctagggtgg aaaggagacc aag 23 30 1234585 玖, Description of the invention < 21〇 > 4 < 211> 18 < 212 > DNA 5 < 213 > Artificial sequence < 22〇 > < 223 > For HURP amplification 10 < 4〇〇 > 4 caacgaaaac agatgctc 18 < 210 > 5 15 <2; ι > 18 < 212 > DNA < 213 > Artificial sequence < 22〇 > 20 < 223 > Amplification for HURP < 4〇〇 > 5 tgagtagc tg at cgag tc 18 31 1234585 发明, Invention Description C Schematic ^^ _ 言 言明 j Figure 1 shows the full length of the human HURP gene nucleotide sequence (sequence identification number: 1), in which the nucleotide sequence complementary to four primers designed according to the present invention is Underlined and shown in bold; 5 Figure 2 shows a restriction map of the vector pET32a (Novagen Inc ·) used in the following examples, where restricted addresses such as mi / I are used to insert the human HURP One full-length nucleotide sequence of one gene; Figures 3A and 3B respectively show HURP and 10 in TCC tissues where N = tumor adjacent tissue, τ = tumor tissue, and 294, 306, 307, 315, 317, 319 , 320, 321, 322, 327, 335, and 337 = patient numbers; Figure 4 shows 5 taken from benign prostatic hypertrophy RT-PCR analysis of human HURP gene expression in prostate samples from patients with dysentery disease15, in which the left half is β-actin (307 bp) and the right half is HURP (370 bp); Nl, N2 , N3, N4, and N5 = 5 benign prostatic hypertrophy (BPH) samples; NC = negative control group; and M represents molecular weight markers showing test-base pairs; and the 5th A and 5B graphs each show ^ ^ RT-PCR analysis of HURP and β-actin in sample 20, where 2, 6, 7, 8, 14, 15, 20, 5, 10, 11, 12, 13, 16, and 18 = patient number ; NC = negative control group; and M indicates molecular weight markers in base pairs. [List of Symbols for the Main Components of the Formula] (None) 32

Claims (1)

Year of the Shirt / I-Month Amendment -f, Patent Application No. 091122555-Tiger Patent Application Application Amendment of Patent Scope Amendment Date · November 1993 1.-A kind of in vitro detection _ Bladder in human body A method for cancer, comprising the following steps: (1) taking a biological sample from an individual suspected of having bladder cancer to detect the expression of a human liver cancer up-regulating protein (HURP) gene, the human HURp gene is The measured performance is a sign of the presence of bladder cancer. 10 2. The method according to the scope of patent application, wherein the bladder cancer is metastatic epithelial cancer (TCC) of the bladder. 3. The method of claim 1, wherein the biological sample comprises cells selected from the group consisting of urinary epithelial cancer cells, bladder cancer cells, or a combination of the two. 15 4. The method of claim 1 in which the biological sample is selected from the group consisting of urine, urinary epithelial biological specimens, blood, plasma, and serum. 5. The method according to item 4 of the patent application, wherein the biological sample is urine. 20 6. The method according to the scope of the patent application, wherein the human HuRp gene has the acid sequence as described in the related serial number: 丨. 7. The method according to item 6 of the scope of patent application, wherein the expression of the human HURP gene is performed by the presence of the mRNA transcript of the human HURP gene in the biological sample. 8. The method according to item 7 of the scope of patent application, in which the human leg P gene 1 1234585 is found and the 范围 N-A to H of sra patent scope exists, by using the following methodology " ^ 被 入 被 T •• Hybridization, Shield Ring Probe Reaction, Polymerase Chain Reaction (PCR), | ^, Reverse Transcription, Polymerase Chain Reaction π ™), Multiple PCR Polymer Chain Reaction, Single Strand Polymorphism, Linkage Chain reaction (LCR), amplification reaction (nasba) mainly based on restriction fragment length polymorphism nuclear sequence, and transcription / regulation amplification reaction (TMA). For example, the method of claim 7 in which the existence of the mRNA transcript of the human hurp gene 10 is performed using rtin-pCD. 1 0. As stated in the method of claim 7 of patent I 巳, wherein the cumulative measurement of the mRNA transcript of the human rib gene is performed by the following steps: 0) total rNA is extracted from the biological sample; (b) introducing the extracted RNA from step (a) to a reverse transcription polymerase chain reaction (rt_PCR) process using at least one primer pair having a forward primer and a reverse primer, each primer having a Nucleic acid sequence will be hybridized to a nucleotide sequence corresponding to sequence identification number: at least J 3 nucleotides of human HURP gene; and (0 to detect if there is a nucleotide sequence identical to or An RT-PCR product complementary to a part of the nucleotide sequence of the human HURP gene has been generated from the RT-PCR treatment in step (b), and the presence of the RT-PCR product is a sign of the presence of bladder cancer. 2 J234585 5 10 15 Scope of patent application]. For example, the scope of application for patents 〖〇 Φ ^ ^ Σ, ， is law, which is used in step (b A the at least one primer pair package '## 2 φ t Has a choice of sequence identification sign. 2 and Column identification number: Gu θ T of the nucleotide sequence of the sequence, the primers, and a sequence with the sequence identification number: 3 and Sequence Office A edit 旒: 5 ... The reverse primer of the glycine sequence. 2. For example, please refer to the method in item 10 of the patent scope, where the at least one primer used in step (b) T pairs with a person — 3 has a corresponding sequence identification,. No .: ^ χϊ of the nucleotide sequence of 4) and a core with a sequence corresponding to the sequence identification number: 5; reverse primer of the acid sequence. 13. The method according to the scope of the patent application] No. 0, in which steps The RT-PCR process in (b) includes a first-amplification reaction using a sub-pair. The first-primer pair has a first-forward primer that has a nucleotide sequence that depends on the identification number: 2 and A first reverse primer with a nucleotide sequence corresponding to the sequence identification number: 3; and a second amplification reaction using a second primer pair, the second primer pair having a Sequence identification number: the second forward primer of the nucleotide sequence of 4 and A second reverse primer with a nucleotide sequence corresponding to the sequence identification number: 5. 14. A method for monitoring bladder cancer in a human individual, comprising the following steps: (1) pair A biological sample from an individual suspected of having bladder cancer was used to detect the expression of a human liver cancer ascending-regulated protein (HURP) gene. The human HURP gene was detected as having a bladder. Characterization of the presence of cancer. 3 20 1234585 The method of applying for patent scope 15. The 14th item of the June patent application, wherein the bladder cancer is the metastatic epithelial cancer (TCC) of the bladder. 16. The method of claim 14 wherein the biological sample package contains cells selected from the group consisting of urinary epithelial cancer cells, bladder 5 cancer cells, or a combination of the two. 17. The method according to the patent application, wherein the biological sample is selected from the group consisting of urine, urinary epithelial biological specimen, blood, plasma, and serum. Ϊ 8. According to the method of applying the patent Nos. Η, ^ τ, and the method, wherein the biological sample is urine 1G. 19. The method as claimed in claim VII, wherein the human visceral p gene has a nucleotide sequence as described in SEQ ID NO: 1. 20. The method according to item 19 of the scope of patent application, wherein the performance of the human population of 15 to 20 is performed by measuring the presence of the mRNA transcript of the human HURP gene in the biological sample. For example, the method of item 2 () in the scope of patent application, wherein the detection of the presence of mRNA transcripts of the human rib gene is performed by using the following methods to v 纟: heterozygous, circular detection Needle reaction, polymerase chain reaction (PCR), nested PCR, reverse polymerase chain reaction (RT-PCR), multiplex PCR yoke reaction_single-strand structural polymorphism, ligase chain reaction Han Ying (LCR), limiting fragment length polymorphic nucleic acid sequence-based amplification reaction (NASBA), and transcription-regulated amplification reaction (TMA). 22 If you apply for the special method of item 2 (), it is the human base 4 1234585. The detection of the existence of the patent thorny miscellaneous mouth of the patent scope—the fufu green 0 mouth, uses RT. -PCR was performed. 2 3. According to the scope of patent application ^; 20 methods of Gudi, in which the mRNA of the human HURP gene Hankou was detected from the presence of six α W green buckles by the following Step 5 is carried out: (a) Total RNA is extracted from the " Xuan biological sample; () The RNA extracted from step (a ') is introduced into one using ^ with a forward primer and a reverse The reverse transcription polymerase chain reaction (RT-PCR) treatment of the primer pairs of the primers, each primer 10 * a ^ sequence will be mixed to-* yes-Schop acid sequence corresponds to the sequence identification number: i of the human HURp gene U nucleotides; and (c) Predict whether or not there is-with a nucleus; the 15 RT-PCR product of which the acid sequence is the same as or complementary to a part of the nucleotide sequence of the human HURP gene has been removed from step (b,) The RT-PCR process is generated. The existence of the Xuan RT-PCR product is a sign of the presence of cancerous tumors. 24. The method according to item 23 of the patent application, wherein the at least one primer pair used in step (b,) includes a sequence identification number 20, which is selected from 2 and a sequence identification number: 4 The forward primer of the nucleotide sequence of the sequence described in 'and-has-selected the reverse primer of the nucleotide sequence of the sequence described in sequence identification number 3 and sequence identification number: 5 25. If applied The method of item 23 of the patent, wherein the at least one primer pair used in step ii) contains a sequence of a nucleotide sequence corresponding to sequence identification 5 1234585, patent application scope number: 4 &檇 向 弓 丨 子, and a reverse primer with a nucleotide sequence corresponding to the sequence identification number: 5. 26. The method of applying for the scope of the patent application No. 23 Xixishi 1 ^ shell, wherein step (b,) The RT-PCR process includes: a first amplification reaction that makes a hi ^ ,, ^ m brother a primer pair, the first primer pair has _ s +, and one has a corresponding sequence identification number: 2 The forward-directed primer of the nucleotide sequence -Has a first reverse primer corresponding to the nucleotide sequence of sequence identification number: 3; and-a second amplification reaction using n sub-pairs, the second primer pair having one having a sequence corresponding to 10 15 20 Judgment and subtraction editing: the second forward primer of the nucleotide sequence of 4 and the second reverse primer of the nucleotide sequence corresponding to the sequence identification number: 5 27. —a DNA sequence, which can be searched from right to right. Eighteen can be selected from the sequence identification number: 2 to sequence identification number: 5 of the sequence described in Nucleic acid sequence. 28.-Species for _- human individual Bladder cancer primer set, which includes-has-selected from the sequence identification number: 2 and sequence identification number: 4 M acid sequence-like forward primers, and-has a selected sequence identification number : 3 and reverse primer of the nucleotide sequence of the sequence described in SEQ ID NO: 5. 1 The primer set according to item 28 of the scope of patent application, wherein the forward primer has a nucleotide sequence as described in sequence identification number: 2. 〇. The primer set of item 28 of the patent application scope, wherein the forward primer has a nuclear sequence as described in the sequence identification number: 4. 3! • The primer set of item 28 in the scope of the patent application, wherein the reverse primer smells like a mu acid sequence as described in the sequence identification number: 3. 6 1234585 Scope of patent application 32. The primer set of item 28 in the scope of patent application, wherein the reverse primer has the nucleotide sequence as described in sequence identification number: 5. 33 · —A test kit for detecting bladder cancer in a human individual, which contains at least one designed for a human HURP gene having a gallic acid sequence corresponding to sequence 5 identification number: 1 Primer pair, the at least one primer pair includes -has-selected from the sequence identification number: 2 and sequence identification number: the forward sequence of the nucleotide sequence of the nucleotide sequence and I has-selected from the sequence identification number: 3 and the reverse primer of the nucleotide sequence of the sequence described in the sequence identification number: 5. 1034. The inspection kit of the 33rd patent application scope includes: a first forward primer corresponding to a sequence identification number: 2 amino acid sequence; and a first primer having a corresponding sequence identification number: 3 The first primer pair of the first reverse primer of the nucleotide sequence and the second forward primer of the nucleotide sequence having a sequence corresponding to the sequence identification number: 4 and 15 have a sequence identification number corresponding to the sequence number: The second primer pair of the second reverse primer of the 5 nucleotide sequence. 7