RNA SNP Detection Method With Improved Specificity Based on Dual-competitive-padlock-probe

Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.

Application of Abnormal Touchdown PCR with High Degeneracy Primer in Amplification of Large-Family Genes

[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Clinicopathologic significance of HER-2/neu protein expression and gene amplification in gastric carcinoma

AIM:To study the HER-2/neu protein expression and gene amplification in gastric carcinoma and their relation.METHODS:One hundred and forty-five formalin-fixed and paraffin-embedded tumor tissue samples from Chinese gastric carcinoma patients were studied with immunohistochemistry(IHC)and fluorescence in situ hybridization(FISH)methods.Clinicopathologic data about all patients were collected.RESULTS:The levels of HER-2 3+,HER-2 2+and HER21+were measurable in 6.9%,8.3%and 17.2%of the samples,respectively.No HER-2 was stained in 67.6% of the samples.FISH showed that HER-2 gene was amplified in 18 samples,10 HER-2 3+samples,5 HER-2 2+samples,and 3 HER-2 1+samples with IHC staining.HER-2 status was not correlated with the sex and age of patients,and tumor size,location or differentiation,but with the depth of invasion,TNM stage,lymph node and distant metastasis as well as histopathological classification of gastric cancer(P<0.05).CONCLUSION:All samples with IHC as HER-2 expression should be analyzed with FISH.Detection of HER-2 gene amplification can assess the malignant biological behaviors and prognosis of gastric cancer.

EGFR and HER2 expression in advanced biliary tract cancer

AIM:To analyze the pathogenetic role and potential clinical usefulness of the epidermal growth factor receptor(EGFR)and the human epidermal growth factor receptor 2(HER2)in patients with advanced biliary tract cancer(BTC). METHODS:EGFR and HER2 expression was studied in biopsy samples from 124 patients(51%women; median age 64.8 years),with advanced BTC diagnosed between 1997 and 2004.Five micrometers sections of paraffin embedded tissue were examined by standard, FDA approved immunohistochemistry.Tumors with scores of 2+or 3+for HER2 expression on immunochemistry were additionally tested for HER2 gene amplification by fluorescence in situ hybridisation(FISH).RESULTS:34/124 patients(27.4%)had gallbladder cancer,47(37.9%)had intrahepatic BTC and 43(34.7%)had extrahepatic or perihilar BTC.EGFR expression was examined in a subset of 56 samples. EGFR expression was absent in 22/56 tumors(39.3%). Of the remaining samples expression was scored as 1+in 12(21.5%),2+in 13(23.2%)and 3+in 9(16%), respectively.HER2 expression was as follows:score 0 73/124(58.8%),score 1+27/124(21.8%),score 2+ 21/124(17%)and score 3+4/124(3.2%).HER2 gene amplification was present in 6/124,resulting in an overall amplification rate of 5%. CONCLUSION:Our data suggest that routine testing and therapeutic targeting of HER2 does not seem to be useful in patients with BTC,while targeting EGFR may be promising.

Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology

AIM:To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).METHODS:120 cases of primary gastric carcinomas and 45 matched lymph node metastases from patients with full clinicopathological features were mounted onto multiple-punch and single-punch tissue microarrays,respectively,and examined for HER2 overexpression and gene amplification by IHC and CISH.RESULTS:Twenty-four tumors (20%) expressed HER2 immunohistochemically.An IHC score of ≥ 2+ was observed in 20 tumors (16.6%).HER2 amplification was detected by CISH in 19 tumors (15.8%) and in their matched lymph node metastases.A high concordancerate was found between HER2 positivity (as detected by IHC) and HER2 gene amplification (as detected by CISH),since 19 of the 20 IHC positive cases were amplified (95%).All amplified cases had 2+ or 3+ IHC results.Amplification was associated with intestinal phenotype (P < 0.05).No association with grading,staging or survival was found.CONCLUSION:In gastric cancer,HER2 amplification is the main mechanism for HER2 protein overexpression and is preserved in lymph node metastases.

ANALYSIS OF C-HA-RAS GENE AMPLIFICATION AND MUTATION IN LARYNGEAL CARCINOMA

In order to study the altered molecular events during laryngeal carcinogenesis and elucidate the role of Haras oncogene amplification and mutation. we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleotide hybridization. We analyzed the mutational status of codon 12 of Haras in 22 laryngeal carcinomas and 10 normal tissues. and found that 7 of 22 laryngeal carcinomas contained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues revealed mutation. Moreover. no amplification was found in cancers when compared to the normal. Ourfindings indicated that the activated Ha-ras gene existed in laryngeal carcinoma. and activation of the Haras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

The RACE amplifying and sequence analyzing of secreted aspartic proteinase gene SA76 of Trichoderma harzianum

Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA （28S and 18S） were observed in agarose electrophoresis. By joining the 3＇end sequence with the known SA76 EST from cDNA library of T. harzianum, a full-length cDNA sequence of 2019bp was obtained, whose open reading frame contained 1593bp, a stop codon TAA, a 5＇untranslated region （5＇UTR） of 266bp, a 3＇untranslated region （3＇UTR） of 201bp, and poly （A） 29 encoded a protein of 530 amino acids, had a signal peptide. T. harzianum shared 53% identity of secreted aspartic proteinase gene with G. zeae, 37% with N. crassa and 36% with C. globosum. The full-length cDNA sequence of secreted aspartic proteinase gene from T. harzianum was cloned for the first time by using BD SMART RACE technique, which provides a foundation to obtain and validate functional genes of T. harzianum.

Feedforward Variable Structural Proportional-Integral-Derivative for Temperature Control of Polymerase Chain Reaction

To track the rapidly changing temperature profiles of thermal cycling of polymerase chain reaction （PCR） accurately, an innovative feedforward variable structural proportional-integral-derivative （FVSPID） controller was developed. Based on the step response test data of the heat block, a reduced first order model was estabfished at different operating points. Based on the reduced model, the FVSPID controller combined a feedforward path with the variable structural proportional-integral-derivative （PID） control. The modified feedforward action provided directly the optimal predictive power for the desired setpoint to speed up the dynamic response. To cooperate with the feedforward action, a variable structural PID was applied, where the P mode was used in the case of the largest errors to speed up response, whereas the PD mode was used in the case of larger errors to suppress overshoot, and finally the PID mode was applied for small error conditions to eliminate the steady state offset. Experimental results illustrated that compared to the conventional PID controller, the FVSPID controller can not only reduce the time taken to complete a standard PCR protocol, but also improve the accuracy of gene amplification.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

Amplifi cations of NCOA3 gene in colorectal cancers in a Chinese population

AIM: To investigate the copy number variation of NACO3 gene in colorectal cancer (CRC) and its correlation with tumor progression. METHODS: A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection. Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues. RESULTS: Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlatedwith tumor progression (χ2 = 6.42, P = 0.0112). Moreover, there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P = 0.0023). Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs 2.71 ± 0.64, P < 0.01). CONCLUSION: Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.

Rapid response of brain metastasis to crizotinib in a patient with KLC1-ALK fusion and MET gene amplification positive non-small cell lung cancer:a case report

Non-small cell lung cancer(NSCLC) ranks as the leading cause of cancer-related death in the world. Brain metastasis(BM) is a common complication of NSCLC, with 25%–40% of patients developing BM during the course of the disease. A significant strategy of local disease control in the central nervous system is radiation therapy. With the development of precision medicine,the concept of treating lung cancer BM has gradually changed. In this case, we performed a surgical procedure to obtain enough tumor tissue for the detection of the target gene and other related experiments after the patient was informed. Finally, we found that the patient had both hepatocyte growth factor receptor(MET) gene amplification and kinesin light chain 1-anaplastic lymphoma kinase fusion(KLC1-ALK) through next-generation sequencing and showed sensitivity to the targeted therapy of crizotinib. The patient exhibited good response. Our case was successful and underwent targeted therapy with the guidance of precise diagnosis.

One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Gene Rearrangement and Mammary Tumor in p53 Transgenic Mice

Gene mutation, rearrangement and amplification are frequently observed in mammary tumors. It is interesting to study if these genetic alterations are involved in DMBA-induced mammary tumors in 172￣(Arg-Leu) mutant p￣(53) transgenic mice. The results of this study suggest that rearrangement of PCNA and H-ras contributed to the production of mammary tumors. No apparent gene amplification, however, was observed in these mammary tumors though several fold higher overexpression of cyclin D1 vs. normal was deteeted. The higher the expression level of 172￣(Arg-Leu) mutant p￣(53) in mammary gland of transgenic mice, the later the mammary tumor appeared in these mice. The 172￣(Arg-Leu) mutant p￣(53) in these mammary tumors behaved similarly to in vitro system.

Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.

A STUDY OF C-MYC ONCOGENE EXPRESSION AND AMPLIFICATION IN COLORECTAL CANCER

Expression of c-myc oncogene transcripts in colorectal neoplasia was studied in paraffin embedded tissue sections from 25 patients undergoing surgery and from the rectal carcinoma cell line HR-8348 by using in situ hybridization,and its amplification was investigated in tumor and normal mucosa tissue from 25 coloproctomy samples by slot blot hybridization. Overexpression of this gene was seen in 78% (7/9) of the benign adenomas and 91% (20/22) of the malignancies sampled. There was no significant correlation between overexpression and the histologic type or grade, and no significant relationship between the level of expression and clinical stage was found, although overexpression was apparently more common in tumors with metastasis. Amplification of the gene was found in 0 of 4 benign adenomas and 7 of 22 malignancies. No obvious correlation was found between amplification and histological type or grade, though amplification was more frequent in tumors with metastasis. Amplification was also found in 2 adenomas with malignant change. The results suggest that multiple factors are involved in the progression of colorectal cancer , and in situ hybridization with a nonradiolabeled probe is useful in the detection of gene expression.

Study on the protein expression and amplification of HER2 gene in gastric cancer

Objective: The aim of the study was to investigate the human epidermal growth factor receptor 2(HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods: Immunohistochemistry(IHC) and fluorescence in situ hybridization(FISH) technique were used to detect HER2 gene amplification and expression of HER2 protein in 442 cases of gastric mixed carcinoma. Results: The expression rate of HER2 protein was 41.2%(182/442): the HER2 protein expression IHC 3+ extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442); the HER2 protein expression IHC 2+ extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442); the HER2 protein expression IHC 1+ extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion: HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplification is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic effect of anticancer drugs.

Analysis of the Relationship between Genome Diversity and Adult Survive Rate of Botryllus Schlosseri by AFLP

Objective: The self cross colonial prochordate, Botryllus schlosseri ( B.schlosseri ) occupy a key phylogenetic position in the evolution of vertebrates. To clarify the relationship of genome diversity and survive rate, five generations of B. schlosseri was investigated by amplified fragment length polymorphism (AFLP). Methods: AFLP markers are extremely sensitive to even small sequence variation, using PCR and high resolution electrophoresis to examine restriction fragments. Results: AFLP polymorphism was high in the parent and lower in its F1, F2, F3 and F4. Each primer combination generated from 80 to more than 120 bands, of which average 25.85% polymorphic loci in parent, 15.79% polymorphic among F1, 9.16% and 5.58% in F2, F3. The AFLP markers were transmitted from F1 to F2, F3 and F4 and inherited, segregated in expected Mendelian ratio. However, some of the markers were lost in F2, F3 and F4 while it disappeared in their mother. In addition, gene mutation new loci and lost loci among F1, F2, F3 and F4 were observed. These special fragments were cloned and sequenced. Then, the genomic DNA was analyzed by Southern hybridization with the probes from these specific fragments and the mechanism of gene mutation was clarified. Conclusion: These results suggest that there are high frequency of polymorphic loci and mutation in genome of B. schlosseri. Gene deletion or low diversity may be the reason for high rate of death of the offspring of inbred laboratory reared strains.

Detection of Listeria monocytogenes in Dairy Food by Loop-mediated Isothermal Amplification(LAMP)

[Objective] This study aimed to detect the reliability of LAMP method for detecting Listeria monocytogenes in dairy food. [Method] Based on the sequence of hlyA gene encoding listeriolysin O in Listeria monocytogenes, the LAMP method was established for detecting Listeria monocytogenes in dairy food. [Result] The es- tablished LAMP rapid detection method has .high specificity and sensitivity, which are equivalent to those of real-time fluorescent quantitative PCR. The detection re- sults of Listeria monocytogenes in dairy food by established LAMP method were completely consistent with those by bacterial isolation method. [Conclusion] The de- tection results of Listeria monocytogenes by LAMP method can be directly identified by naked eye, so the established LAMP method was suitable for the detection of Listeria monocytogenes in dairy food in emergency situations.

The role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis in lung cancer with c-Met amplification

Objective This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.Methods In vitro, H2228, H1993, and A549 cells were treated with crizotinib. The inhibition of proliferation was quantitated by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. Apoptosis was quantified by flow cytometry. Expression of key proteins of the HGF/c-Met signaling pathway was examined by western blotting. In vivo, H1993 and A549 tumor cell xenograft models were established. Immunohistochemical analysis was used to determine protein expression of HGF and c-MET and the amount of phospho-c-MET(p-c-Met). Real-time quantitative polymerase chain reaction(PCR) was applied to examine the messenger RNA(m RNA) expression of c-MET and serine/threonine protein kinase(AKT). The expression and activation of the key proteins were evaluated by western blotting.Results In vitro, the growth of H1993, H2228, and A549 cells was inhibited after crizotinib treatment for 72 h. Apoptotic rates of H1993 and H2228 cells increased with the crizotinib concentration and exposure time. In vivo, the growth-inhibitory rate of crizotinib for H1993 xenografts was 72.3%. Positive expression rates of HGF and c-MET in H1993 xenografts were higher than those in A549 xenografts; the p-c-MET amount was the largest in H1993 xenograft control but the lowest in the H1993 xenograft with crizotinib treatment. The m RNA expression levels of c-MET and AKT in H1993 xenografts were higher than those of A549 xenografts. The protein levels of c-MET, AKT, and extracellular regulated protein kinases(ERK) in H1993 xenografts were higher than those in A549 xenografts; the p-AKT amount was higher in H1993 xenograft control than in A549 xenografts; the largest amount of p-c-MET was detected in H1993 xenograft control; the amount of p-ERK was the lowest in the H1993 xenograft with crizotinib treatment.Conclusion The HGF/c-Met signaling pathway may mediate crizotinib-induced apoptosis and inhibition of proliferation of lung adenocarcinoma cells.