Specific PCR Identification of Hibisci Cortex and Its Adulterants Based on DNA Signature Sequence Tags / 中国实验方剂学杂志

ObjectiveUncommon medicinal herbs are valuable medicinal resources， but their identification is a difficult problem in Chinese medicine due to their particularity and complexity. It is， therefore， urgent to establish a method for the identification of uncommon medicinal herbs. In this study， DNA signature sequence （DSS） tags were used to establish a specific polymerase chain reaction （PCR） identification method for Hibisci Cortex， the origin plant of Hibisci Cortex， and its adulterants. MethodThe candidate DSS tags were obtained from the chloroplast genome sequence analysis， and the DSS tags were verified by DNA sequencing. The specific identification primers for H. syriacus were designed based on the obtained reliable DSS tags. The PCR reaction conditions were optimized， and the tolerance and feasibility were investigated. ResultA DSS tag for identification of H. syriacus was obtained from the comparison of sequencing results of the amplified products with DSS， which revealed the distinguishing characteristics of Hibisci Cortex and its adulterants. A pair of specific primers for H. syriacus was designed according to the DSS tag. After PCR amplification and gel electrophoresis with the primers， a single bright band of about 270 bp was observed from H. syriacus， which did not appear in the four adulterants. ConclusionA DSS tag obtained in this study can be used to identify H. syriacus. The specific primers designed based on this DSS tag can accurately and simply identify the original plant of Hibisci Cortex and its adulterants， which provides a new method and idea for the molecular identification of genuine and counterfeit products of Hibisci Cortex.

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction（PCR） method to accurately discriminate the crude materials of Murrayae Folium et Cacumen， Murraya exotica and M. paniculata. MethodBased on the difference in chloroplast genome sequences of M. exotica and M. paniculata， species-specific identification primers P03 and P04 of M. exotica and M. paniculata were designed according to single nucleotide polymorphism （SNP） on the chloroplast genome. A multiplex allele-specific PCR identification method was established for the identification of M. exotica and M. paniculata following the optimization of annealing temperature， number of cycles， and primer concentration ratio. The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model. ResultIn this multiplex allele-specific PCR identification method， about 330 and 230 bp of specific fragments were amplified from DNA templates of M. exotica and M. paniculata， respectively， under the following conditions：cycle number of 31， annealing temperature of 60 ℃， and primer concentration ratio of P03 and P04 of 1∶2. Consistent results were obtained for samples from different sources. ConclusionThe multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen， which can be used for the simultaneous identification of M. exotica and M. paniculata by the length of fragments in a single identification assay.

Correlation between the Polymorphism of Coagulation-Related Genes and Lower Extremity Deep Venous Thrombosis / 法医学杂志

Objective To investigate the correlation between the polymorphism of 4 coagulation-related genes, rs1799963 （coagulation factor V gene Leiden）, rs6025 （prothrombin gene G20210A）, rs1042579 （thrombomodulin protein gene c.1418C>T） and rs1801131 （methylenetetrahydroflate reductase gene） and lower extremity deep venous thrombosis （LEDVT）. Methods The 4 genotypes mentioned above of 150 LEDVT patients and 153 healthy controls were detected by the kompetitive allele specific polymerase chain reaction （KASP）, then related blood biochemical indicators were collected, binary Logistic regression was established to screen the independent risk factors of LEDVT, and the correlation between polymorphism of 4 coagulation-related genes and LEDVT and its indicators under different genetic modes after adjusting confounding factors were analyzed. Results Five variables, D-dimer, fibrinogen degradation product, homocysteine, sex and age might be the risk factors of LEDVT. These variables were put into 4 genetic inheritance models, and adjusted in binary Logistic regression. The results suggested that the mutations of rs1042579 were correlated with LEDVT under dominant inheritance mode. Conclusion The gene polymorphism of rs1799963, rs6025 and rs1801131 has no significant correlation with the formation of LEDVT. The gene polymorphism of rs1042579 plays a role under dominant inheritance mode, and might be an independent risk factor for formation of LEDVT.

Construction of SNP-STR Multiplex Amplification System with Genetic Markers and Its Forensic Application / 法医学杂志

Objective To select and develop a SNP-STR multiplex amplification system with genetic markers compatible with current STR databases. To understand its genetic polymorphisms in Sichuan Han population and its application value in DNA mixture analysis. Methods Based on the STR genetic markers in commercial kits, SNPs adjacent to these STR markers were selected to be SNP-STR genetic markers. A SNP-STR multiplex amplification system with genetic markers based on allele-specific amplification was constructed using allele-specific amplification primers. The genetic polymorphism of the system in the Sichuan Han population was investigated and the efficiency of systems with different numbers of loci to detect the two individual DNA mixture samples was evaluated. Results An allele-specific multiplex amplification system constituted of 13 SNP-STR genetic markers was selected and constructed. In Sichuan Han population, the heterozygosity of each locus ranged from 0.76 to 0.88, and the combined discrimination power reached 0.999 999 999 999 999 968. In the analysis of the two individual DNA mixture samples： for single-locus amplification, the genotype of the minor components can still be detected when the mixture ratio reaches 1 000∶1; for multiple loci multiplex amplification, the maximum mixture ratio can reach 500∶1. As the number of loci in the system increased, the detection efficiency of the minor components in the DNA mixture decreased. Conclusion SNP-STR genetic markers have a higher polymorphism than STR. The multiplex amplification system made of SNP-STR genetic markers has a better analysis efficiency for mixed samples than traditional STR multiplex amplification system.

Identification of Corydalis yanhusuo,C. turtschaninovii,C. decumbens by allele-specific PCR / 中国中药杂志

To establish a DNA molecular markers method for identification of Corydalis yanhusuo,C. turtschaninovii and C. decumbens,the mat K,trn G and psb A-trn H sequences of 56 samples from 14 species of C. yanhusuo,C. turtschaninovii,C. decumbens and their related species were obtained by sequencing. The SNP loci were obtained by Bio Edit 7. 2. 2 software. The primers for AS-PCR identification were designed based on the mutation sites,and the conditions of PCR were optimized to identify C. yanhusuo,C. turtschaninovii,and C. decumbens according to the specific bands. The results showed that the amount of template( 0. 6-1 200 ng)and annealing temperature( 42-60 ℃) had little influence on the amplification results,and the number of cycles had much influence on the amplification results. When the number of cycles was 20,the specific bands of 297 bp( mat K),353 bp( trn G) and 544 bp( mat K) were amplified from C. yanhusuo,C. turtschaninovii and C. decumbens,respectively. The method established in this study had a minimum detection limit of 6 ng for C. yanhusuo,60 ng for C. decumbens and less than 0. 6 ng for C. turtschaninovii. Thus,the allelespecific PCR method established in the research can specifically identify C. yanhusuo,C. turtschaninovii,and C. decumbens.

Application of a new GC rich region sequence-specific polymerase chain reaction (SS-PCR) method for detection of C729T SNP / 西安交通大学学报(医学版)

Objective: To develop a sequence-specific polymerase chain reaction (SS-PCR) method for detection of single nucleotide polymorphism (SNP) in GC rich region and analyze the C729T SNP in the exon 3 of estrogen receptor α gene of endometrial cancer (EC). Methods: We selected 22 EC and 42 controls. A new GC rich region SS-PCR was developed. The two internal specific primers, identical to the two single strands of double strand DNA, were designed, and the 3' end of the primer coincided with the SNP locus. The PCR extension was controlled by the 3' end primer and the SNP was determined according to the bands of the extension product. The specificity of the extension reaction was increased by introducing a mismatched base at the third position upstream in the 3' end of the SNP specific primer. This method was used to confirm C729T genotypes in the exon 3 SNP, followed by direct sequencing. Results: The results showed that the SS-PCR was appropriate for genotyping C729T SNP of exon 3 in human ERα gene. Conclusion: A GC rich region SS-PCR method developed can be successfully applied in C729T SNP determination in the exon 3 of estrogen receptor α gene of endometrial cancer.

Specific PCR Method for Identification of Suis Fellis Pulvis and Its Chinese Patent Medicines / 中国实验方剂学杂志

Objective: A polymerase Chain reaction(PCR) identification method for Suis Fellis Pulvis and its Chinese patent medicines was established to provide an example for the identification of animal-derived components in complex components. Method: A PCR identification method was established based on swine derivatives identification primers,the reaction system was optimized,and the established method was investigated and verified. By the established PCR identification method,the swine derivatives of 20 batches of self-made Suis Fellis Pulvis material,19 batches of commercially available Suis Fellis Pulvis and 22 batches of Chinese patent medicines containing Suis Fellis Pulvis were identified. The commercially available Suis Fellis Pulvis material and Chinese patent medicines containing Suis Fellis Pulvis positive products that were amplified PCR were verified by enzyme digestion and sequencing. Result: Totally 20 batches of self-made Suis Fellis Pulvis material and Suis Fellis Pulvis control material could expand the specific identification band of about 212 bp,and there was no bands in bovine and ovine reference, only 5 batches of the 19 batches of commercially available Suis Fellis Pulvis had expanded specific identification bands, 10 batches of 22 batches of Chinese patent medicines containing Suis Fellis Pulvis were detected to have swine derivatives, the Suis Fellis Pulvis control material and the PCR-amplified commercially available Suis Fellis Pulvis material positive products can produce about 200 bp of bands after digestion with Mnl I. The highest similarity between the amplification products sequence of Suis Fellis Pulvis and its Chinese patent medicines, and the GenBank database was Sus scrofa,the consistency was 99%,which conformed to the sequence of swine. Conclusion: The PCR identification method established in this paper can accurately identify the biological origin of Suis Fellis Pulvis and its Chinese patent medicines.

Identification of Pheretima by Multiplex Allele-specific PCR / 中国实验方剂学杂志

Objective: In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market. Besides,the medicinal materials have mostly lost the main identification features, and are difficult to distinguish. Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima. Method: According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR) method for identification was explored and verified in terms of tolerance and feasibility in this study. The specific primers were combined to build multiple PCR systems. An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants. Result: Through the established multiplex PCR reaction system, 366,487,487 and 475 bp of fragments were amplified from DNA templates of P. aspergillum,P. vulgaris,P. guillelmi and P.pectinifera respectively. All of the adulterants were negative by the multiplex PCR assay. The PCR amplification of specific alleles method established in this paper can accurately identify pheretima. Conclusion: The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.

Optimizing a methylation-specific polymerase chain reaction technology / 中国职业医学

OBJECTIVE: To establish a highly specific methylation-specific polymerase chain reaction( MSP) method.METHODS: Bisulphited conversion hypermethylated human genomic DNA and unmethylated human genomic DNA were used as template and p15 gene as the target gene. The Cp G island of p15 gene promoter was predicted by Methprimer software.The methylated and unmethylated primers for polymerase chain reaction( PCR) of methylated and unmethylated DNA templates were designed. The PCR stage of MSP was divided into normal temperature group( methylation primer and unmethylated primer annealing temperature of 58 and 55 ℃,respectively) and elevated temperature group( methylation primer and unmethylated primer annealing temperature were 60 and 57 ℃ respectively). The PCR reaction system was optimized based on the normal temperature group by increasing the concentration of magnesium ions( Mg2 +) and adding dimethyl sulfoxide( DMSO) in the PCR reaction system. RESULTS: Nonspecific amplification products appeared in normal temperature group in the PCR reaction stage of MSP,indicating false positive and false negative reactions. The non-specific amplification was weakened and the specific amplification was equally reduced in the elevated temperature group. The nonspecific amplification disappeared in the optimized PCR system and normal temperature group,and the false positive and false negative reactions were eliminated completely. CONCLUSION: Setting the normal annealing temperature and the concentration of Mg2 +and DMSO is beneficial to ensure the specificity of MSP results.

Identification of Dendrobium huoshanense, Dendrobium officinale and Dendrobium devonianum by multiplex allele-specific polymerase chain reaction / 药学学报

This study was designed to establish a multiplex allele-specific polymerase chain reaction method for simultaneous identification of Dendrobium huoshanense, D. officinale and D. devonianum, which may resolve identification problems of caulis dendrobii. Internal transcribed spacer sequences and trnL-trnF sequences of the Dendrobium species were aligned by BioEdit software, then specific SNPs of the three species were analyzed for designing allele-specific primers and the multiplex allele-specific PCR reaction system was established. The different origin of Dendrobium huoshanense, D. officinale and D. devonianum was amplified and identified by the sizes of respective band. The results showed that 584 bp, 397 bp and 211 bp bands could be amplified by D. devonianum, Dendrobium officinale and Dendrobium huoshanense respectively, when the annealing temperature was 61 ℃ and the number of cycles was 35. The limit of detection (LOD) of D. devonianum and D. huoshanense were both 1.2 ng, while D. officinale was low than 0.24 ng. The detection limit of adulterates in D. devonianum, D. devonianum and D. huoshanense mixture sample was 1%, 1% and 5% respectively. This result suggests that the method of multiplex allele-specific PCR is useful to identify D. huoshanense, D. officinale and D. devonianum is accurate and specific.

Relationship between methylation status of Rap1 GTPase activating protein and colon cancer / 肿瘤研究与临床

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People′s Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43 % (50.35 %), 21.37 % (8.39 %) and 17.43 % (15.71 %) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P60yearsold:36.26%(62.62%)and26.23%(76.42 %);well-differentiated vs. moderately/poorly-differentiated: 21.98 % (40.32 %) vs. 42.74 % (74.20 %); TNM Ⅰ-Ⅱ vsⅢ-Ⅳ: 25.31 % (48.27 %) vs. 36.26 % (75.55 %); all P> 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer.

Identification of Panax ginseng, P. notoginseng and P. quinquefolius admixture by multiplex allele-specific polymerase chain reaction / 中国中药杂志

To achieve a molecular method to identify Panax ginseng, P. notoginseng,P. quinquefolius and their admixture. The ITS,18S and matK sequences of Panax genus were analyzed to develop species-specific SNP marker. Three pairs of species-specific primers were designed to establish a multiplex allele-specific polymerase chain reaction (MAS-PCR) and the samples from different region were tested. The results showed that when the annealing temperature was 60 ℃ and the cycle number was 35, approximately 250, 500,1 000 bp specific band were obtained from P. ginseng, P. notoginseng and P. quinquefolius obtain, respectively. This method could also be used to authentificate admixture samples and could detect 0.5% percent of P. notoginseng or P. quinquefolius adulterated in P. ginseng, or 0.5% percent of P. ginseng or P. quinquefolius adulterated in P. notoginseng. The detect limit of P. ginseng in P. quinquefolius was 0.5% and P. notoginseng in P. quinquefolius was 1%. This results showed that the present method could be used as a promise method to identify Panax ginseng, P. notoginseng, P. quinquefolius and their admixture.

IGF-II gene promoter P3 methylation in salivary pleomorphic adenoma / 实用口腔医学杂志

Objective:To detect the methylation status of insulin-like growth factor Ⅱ(IGF-Ⅱ)gene promoter P3 in salivary pleo-morphic adenoma(SPA).Methods:The methylation of IGF-Ⅱ gene promtor P3 was examined in 26 cases of salivary pleomorphic adenoma with adjacent normal salivary gland tissues,1 0 cases of salivary gland malignant tumor (except malignant pleomorphic ade-noma)and 1 0 cases of normal salivary gland tissue by nested methylation specific polymerase chainreaction(nMSP),1 0 samples(3 SPA and controls,2 malignance and controls)were examined by pyrosequencing.Results:9 out off the 26 SPA showed lower level methylation oevel of IGF-II gene promoter P3.Normal salivary gland and salivary gland malignant tumor tissue did not.Conclusion:IGF-II gene promoter P3 with low level of methylation may play a role in the development of SPA.

Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection / 中华实用儿科临床杂志

Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.

Diagnostic value of promoter methylation and protein expression of plasma RNF180 gene in gastric cancer / 中国肿瘤临床

Objective:To investigate the diagnostic value of the promoter methylation of plasma RNF180 gene and its protein ex-pression for the detection of gastric cancer. Methods:Methylation-specific polymerase-chain reaction (MSP) and enzyme-linked immu-no-sorbent assay (ELISA) were performed to detect DNA methylation and protein expression of the RNF180 gene, respectively. The correlations of DNA methylation and protein expression of the RNF180 gene with the clinico-pathological parameters of gastric carcino-ma were then separately analyzed. Results:MSP showed that the methylation rates of the RNF180 gene were 62.75%and 21.88%in the plasma of patients with gastric carcinoma and healthy volunteers, respectively;this result indicated that the two groups significantly differed (P0.05). Conclusion:The RNF180 gene is expressed at a hypermethylation rate, and the corresponding protein expression level is de-creased in the plasma of individuals with gastric carcinoma. Therefore, RNF180 gene methylation in plasma could be applied to detect microinvasion for the clinical diagnosis of gastric cancer.

A simple, rapid and economic method for detecting multidrug-resistant tuberculosis

OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.

Methylation status of CDH13 gene promoter in colon cancer / 中国医师进修杂志

Objective To detect the methylation status of CDH1 3 gene promoter in colon cancer by using methylation-specific polymerase chain reaction(MSP)technique.Method The tissue specimens from 32 cases of colon cancer(observation group),and 12 cases of normal colon tissues(control group)were examined,the methylation of CDH13 gene promoter was detected by MSP.Result The methylation of CDH13 gene promoter was detected in 19 cases(59.4％,19/32)in observation group,1 case(8.3 ％,1 / 12)in control group,there was statistical significance between two groups(P =0.002).Conclusion Frequency of the methylation of CDH13 gene promoter is apparently higher in colon cancer tissues than that in normal colon tissues,it reveals that CDH13 gene promoter may contribute significantly to the development of colon cancer.

A Consideration of MGMT Gene Promotor Methylation Analysis for Glioblastoma Using Methylation-Specific Polymerase Chain Reaction and Pyrosequencing

BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is currently the most promising predictive marker for the outcome and benefit from temozolomide treatment in patients with glioblastoma, but there is no consensus on the analysis method for assessing the methylation status in the molecular diagnostic field. The objective of this study was to evaluate methylation-specific polymerase chain reaction (MSP) and pyrosequencing methods for assessing MGMT gene promotor methylation of glioblastoma as well as assessing the MGMT protein expression by immunohistochemistry. METHODS: Twenty-seven cases of glioblastoma from the archives at the Department of Pathology Konkuk University Hospital were selected. MGMT promoter methylation was evaluated by MSP and the pyrosequencing methods. The MGMT expression was also measured at the protein level by immunohistochemistry. RESULTS: Overall, MGMT hypermethylation was observed in 44.4% (12/27 cases) of the case of glioblastoma using either MSP or pyrosequencing. The concordant rate was 70.3% (19/27 cases) between MSP and pyrosequencing for MGMT methylation. There was no correlation between MGMT methylation and the protein expression. No significant differences in progression free survival and overall survival were seen between the methylated group and the unmethylated group by using either MSP or pyrosequencing. The status of the MGMT protein expression was correlated with progression free survival (p=0.026). CONCLUSIONS: In this study the concordance rate between MSP and the pyrosequencing methods for assessing MGMT gene promotor methylation was relatively low for the cases of glioblastoma. This suggests that more reliable techniques for routine MGMT methylation study of glioblastoma remain to be developed because of quality control and assurance issues.

Role of DNA methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma / 中华临床营养杂志

Objective To study the methylation status of the promoter region of several tumor suppressor genes in p53-Bax mitochondrial apoptosis pathway and its role in cholangiocarcinoma. Methods The hypermethylation of the promoter region of tumor suppressors death-associated protein kinase (DAPK), p14, and target of methylation-associoted silencing-1 (TMS1/ASC) were detected by methylation-specific PCR. P53 gene status (exon 5-8 ) were examined by automated sequencing. The relationship between gene mutations and the biological behaviors of cholangiocarcinoma was analyzed. Results Methylation existed in at least one promoter region of tumor suppressor gene in the tumor tissues of 24 patients (66. 7% ). The frequencies of tumor suppressor gene methylation in cholangiocarcinoma were: p14 24%, DAPK 30. 6%, and TMS1/ASC 36. 1%. The frequencies of tumor suppressor gene methylation in the adjacent tissues were: TMS1/ASC 8.3% and DAPK 5.6%. DNA sequencing showed p53 gene mutation was found in 22 of 36 patients (61.1% ), and p53 gene mutation combined with the methylation of tumor suppressor was found in 14 (38.9%) patients, which was significantly correlated with pathologic biology, invasion, and differentiation ( P ＜ 0.05 ). The 1-year, 2-year, and 3-year survival rates were significantly higher in tumor-suppressing genes methylation group ( n = 4) (70%, 43 %, and 28%, respectively)than those in p53 gene mutation group (n = 14) (28%, 5%, and 0%, respectively) (χ2 =9. 060, P =0.03).Conclusions Promoter hypermethylation of p53-Bax mitochondrial apoptosis pathway is a common epigenetic event in cholangiocarcinoma. Although the methylations of TMS1/ASC and DAPK genes in the adjacent tissues are relatively low, they may be informative for the early detection of cholangiocarcinoma. P53 gene mutation combined with the methylation of tumor suppressor may be related with the pathologic biology of cholangiocarcinoma, making the latter trend to be with high malignancy and poor prognosis.

Rapid ABO Genotyping Using Whole Blood without DNA Purification / 대한진단검사의학회지

BACKGROUND: ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification. METHODS: A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method. RESULTS: The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies. CONCLUSIONS: This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.