Hypermethylation Analysis of p16INK4a and p15INK4b Promoters in Chronic Lymphocytic Leukaemia Patients and Normal Individuals

@#Introduction: Chronic Lymphocytic Leukaemia (CLL) is a common type of leukaemia in persons of predominantly European descent but is rare in the Asian population. Disparities in CLL incidence among people of Asian and European descent may be related to the genetic make-up of the two different populations. Hypermethylation event might be one of the silencing mechanisms that inactivate the tumour suppressor genes in CLL. The aim of this study was to determine the hypermethylation status of p16INK4a and p15INK4b among CLL patients and normal individuals. Materials & Methods: A total of 25 CLL patients and 25 normal individuals were recruited for this study and their genomic DNA were extracted from the peripheral blood. The hypermethylation status of p16INK4a and p15INK4b were determined using Methylation Specific-PCR (MS-PCR) whereas DNA sequencing method was applied to selected samples for validation of the MS-PCR results. We also evaluated the association between hypermethylation of these genes with the clinical and demographic characteristics of each group of subjects. Results: Among the CLL patients, p15INK4b partialmethylation occurred in 6 (24%) subjects while methylation occurred in 1 (4%) subject. All the remaining patients were unmethylated at p15INK4b. All the samples showed unmethylation at p16INK4a. Statistically significant associations were found between p15INK4b hypermethylation with the presence of CLL (p=0.01) and with race (p=0.02). Conclusion: Further study using a larger sample size is warranted to explore the significance of DNA methylation incidence among the CLL patients of the Malaysian population. Hence, we suggest that hypermethylation at p15INK4b has a huge influence that kick-starts CLL disease among Malaysians and MS-PCR technique is applicable to be used in methylation study.

Clinicopathologic analysis of benign and malignant ovarian goiter / 中国癌症杂志

Background and purpose:Struma ovarii is a rare tumor, especially with extraovarian spreading. The study aimed to investigate the clinical and pathological features, diagnosis and differential diagnosis of ovarian goiter. Methods:Clinical and pathological features of 14 cases of benign and malignant ovarian goiter were observed. Immunohistochemical EnVision staining, PCR-DNA sequencing and review of related literature were also used. Results:In 14 cases of benign and malignant ovarian goiter, the average age of onset is 45.6 years (18-71 years old), and pelvic tumor is the main clinical manifestation. According to the related literature of diagnostic criteria, 12 cases are struma ovarii, which is consisted of hyperplasia of the thyroid tissue under microscopic examination, 1 case is malignant struma ovarii, which is papillary thyroid carcinoma by microscopic presentation, and 1 case is highly differentiated follicular carcinoma of ovarian origin (HDFCO), which is histological benign by microscopic presentation, but is malignant by biological behavior. Conclusion:Struma ovarii is a rare ovarian mondemal teratoma, with low rate of malignant change and beyond ovarian lesions disseminated microscopic histological benign struma ovarii is lower incidence, which has unique clinical and pathological features. Comprehensively considering the related literatures, this study indicates that the disease is in accordance with HDFCO. Struma ovarii prognosis is good, and should be differentiated from carcinoid and granular cell tumor.

Prevalence of Human Papillomavirus Genotypes in Routine Pap Smear of 2,562 Korean Women Determined by PCR-DNA Sequencing

The infections by human papillomaviruses (HPVs) are clearly associated with the subsequent development of cervical cancer. In this study, HPV genotype distribution and prevalence were detected in Korean women from January to December 2008 using PCR-DNA sequencing. A total of 2,562 cervical samples from Korean women having routine Pap smear cytology screening were used. HPV DNA was extracted from cervical swab samples and amplified by PCR in L1 region of HPV. HPV DNA was detected in 23.2% and 65.5% from the groups of normal and abnormal Pap cytology, respectively. The prevalence of high-risk types of HPV had the highest frequency in the <30 year-olds' group (50.6%). The prevalence of HPV in normal, ASCUS, LSIL and HSIL groups was 23.2%, 58.1%, 96.3% and 97.0%, respectively. Moreover, the frequencies of the high-risk types of HPV were 16.2% in the normal Pap cytology, 44.7% in the ASCUS, 76.1% in the LSIL and 94.1% in the HSIL groups. The prevalence of the high-risk types of HPV increased in proportion to the severity of the cytological classification. In the HSIL group, HPV type 16 was the most frequently found at 32.4%, followed by types 58, 53 and 33 at 17.6%, 14.7% and 11.8%, respectively. HPV type 82 was found in 5.6% of the HSIL group and was not detected in the normal Pap cytology group. The frequency of high-risk type of HPV 82 is firstly reported in Korean women. This finding could be an informative basis for the development of future HPV vaccination strategies in Korean women.

Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR

Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.

Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR

Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.

The Progress and Application of Recombinant PCR / 中国生物工程杂志

Recombinant PCR applies to fulfill gene recombination by PCR thermal reaction.Over the twenty years,it has branched into three characteristic strategies:splicing by overlapping extension(SOE),jumping polymerase chain reaction(JPCR)and DNA shuffling.Recently,the technique aimed with exploiting natural source of different allele genes is developing up on simplification of experimental procedure,on trap for mutation and variation,and on highthroughput screening with technology of surface display and fluorescent probe.The recombinant PCR is increasesing value in broad range from biological basic research to bioengineering study.

The Development of Rapid PCR / 中国生物工程杂志

Polymerase chain reaction (PCR) is one of the common techniques in molecular biology, which can amplify nucleic acids through the cycle of denaturation, annealing and extension. Based on the principle of common PCR, rapid PCR is to realize the amplification of nucleic acids in less time without affecting the specificity, sensitivity and fidelity of the reaction. A lot of research work in this field has been going on in recent years. This article will make a review of the development of rapid PCR with emphases on the improvement of DNA polymerase, the choice of additives and the improvement of thermocyclers.

DNA Shuffling of Vitreoscilla Hemoglobin / 中国生物工程杂志

To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.

Hepatitis G virus in hemodialysis patients and gene alignment analysis / 中华肾脏病杂志

Objective To evaluate the prevalence and clinic relevance of hepatitis G virus(HGV)infection in maintenance hemodialysis patients. Methods Reverse-transcription(RT) nested polymerase chain reaction(PCR)was used to detect HGV in 50 HD patients. The prevalence of HGV infection, their relationship with risk factors, liver function and HBV, HCV infection were investigated. Results HGV RNA was found in 14 percent of the HD patients (7 of 50), as compared with none of health blood donors(0 of 20, P

p53 Gene Mutations in Bladder Cancer (I): Comparative Analysis by Immunohistochemistry and DNA Sequencing / 대한비뇨기과학회지

Background. p53 gene mutations are known to play an important role in the progression of bladder cancer. Immunohistochemistry (IHC) has been used routinely to analyze p53 gene mutations by identifying nuclear overexpression of p53 protein. However, the accuracy and value of IHC as a marker of p53 gene mutation has been questioned. Methods. In this study of 35 bladder transitional cell carcinoma, we compared results of IHC staining with those of polymerase chain reaction (PCR) of exons 5 to 8 of p53 gene, followed subcloning of PCR products, and DNA sequencing analysis. Results. On IHC staining, 12 bladder tumors (37.5%) showed overexpression of p53 as defined by nuclear staining of 10% or more of tumor cells. On DNA sequencing analysis, 11 out of 32 cases (34.3%) showed point mutations in one or more exons of p53 gene. The results of IHC were concordant with that of DNA sequencing in 84.3% of cases. The sensitivity and specificity of detecting p53 mutations by IHC were estimated to be 81.8% and 90.5%, respectively. Conclusion. When properly used, IHC is a highly sensitive and specific, and clinically useful method to detect p53 gene mutations in bladder cancer.

A STUDY OF HUMAN MITOCHONDRIAL DNA POLYMORPHISM REGION SEQUENCING / 中国法医学杂志

By means of Polymerase chain reaction (PCR), the mitochondrial DNA polymorphous region from the base number 15997 to 16401 was amplified. After purification,the amplified fragment was directly Sequenced on automatic .DNA sequencer and accurate results was obtained. Compared with the former reports, there are base divergence between the results of this paper and sequence of Caucasian. Changes of base in six samples were observed.The numbers of base change are 3 to 6. The maternal inheritance of the mitochondrial genome makes mtDNA sequencing via PCR an attractive tool for testing whether individuals are maternally related. The method can also be used for studying of race inheritance and molecular evolution. The method is rather simpler and quicker than the former methods.