Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR / 生物工程学报

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.

Cerebrospinal fluid challenges for the diagnosis of herpes simplex infection in the central nervous system / Desafios do exame do líquido cefalorraquidiano para o diagnóstico de infecção por herpes simplex no sistema nervoso central

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.

Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.

Prevalence of mutations in HBV DNA polymerase gene associated with nucleos(t)ide resistance in treatment-naive patients with Chronic Hepatitis B in Central China

Abstract Objective There are a lot of disagreements in the studies on hepatitis B virus (HBV) DNA polymerase mutation rate associated with nucleos(t)ide analogues (NAs) in treatment-naive chronic hepatitis B (CHB) patients. This is the first study aimed to investigate the prevalence of spontaneous HBV resistance mutations in Central China. Methods This study included treatment-naive patients with CHB from June 2012 to May 2015 receiving care at the Institute of Liver Disease in Central China. All patients completed a questionnaire covering different aspects, such as family medical history, course of liver disease, medication history, alcohol use, among others. Mutations in HBV DNA polymerase associated with NAs resistance were detected using INNO-LiPA assay. Results 269 patients were infected with HBV genotype B (81.4%), C (17.9%), and both B and C (0.7%). Mutations in HBV DNA polymerase were detected in 24 patients (8.9%) including rtM204I/V (n = 6), rtN236T (n = 5), rtM250V (n = 2), rtL180M (n = 2), rtT184G (n = 1), rtM207I (n = 1), rtS202I (n = 1), rtM204V/I & rtL180M (n = 5), and rtM204I & rtM250V (n = 1). Conclusion Spontaneous HBV resistance mutations in HBV DNA polymerase were found in treatment-naive patients with CHB in Central China. These findings suggest that we should analyze HBV DNA polymerase resistance mutation associated with NAs before giving antiviral therapy such as lamivudine (LAM), adefovir (ADV), and telbivudine (LdT).

Long-Term Outcomes and Dynamics of Mutants Associated with Lamivudine-Adefovir Rescue Therapy in Patients with Lamivudine-Resistant Chronic Hepatitis B

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.

No CAG repeat expansion of polymerase gamma is associated with male infertility in Tamil Nadu, South India.

Mitochondria contains a single deoxyribonucleic acid (DNA) polymerase, polymerase gamma (POLG) mapped to long arm of chromosome 15 (15q25), responsible for replication and repair of mitochondrial DNA. Exon 1 of the human POLG contains CAG trinucleotide repeat, which codes for polyglutamate. Ten copies of CAG repeat were found to be uniformly high (0.88) in different ethnic groups and considered as the common allele, whereas the mutant alleles (not -10/not -10 CAG repeats) were found to be associated with oligospermia/oligoasthenospermia in male infertility. Recent data suggested the implication of POLG CAG repeat expansion in infertility, but are debated. The aim of our study was to explore whether the not -10/not -10 variant is associated with spermatogenic failure. As few study on Indian population have been conducted so far to support this view, we investigated the distribution of the POLG CAG repeats in 61 infertile men and 60 normozoospermic control Indian men of Tamil Nadu, from the same ethnic background. This analysis interestingly revealed that the homozygous wild type genotype (10/- 10) was common in infertile men (77% - 47/61) and in normozoospermic control men (71.7% - 43/60). Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis.

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

No CAG repeat expansion of polymerase gamma is associated with male infertility in Tamil Nadu, South India.

Mitochondria contains a single deoxyribonucleic acid (DNA) polymerase, polymerase gamma (POLG) mapped to long arm of chromosome 15 (15q25), responsible for replication and repair of mitochondrial DNA. Exon 1 of the human POLG contains CAG trinucleotide repeat, which codes for polyglutamate. Ten copies of CAG repeat were found to be uniformly high (0.88) in different ethnic groups and considered as the common allele, whereas the mutant alleles (not -10/not -10 CAG repeats) were found to be associated with oligospermia/oligoasthenospermia in male infertility. Recent data suggested the implication of POLG CAG repeat expansion in infertility, but are debated. The aim of our study was to explore whether the not -10/not -10 variant is associated with spermatogenic failure. As few study on Indian population have been conducted so far to support this view, we investigated the distribution of the POLG CAG repeats in 61 infertile men and 60 normozoospermic control Indian men of Tamil Nadu, from the same ethnic background. This analysis interestingly revealed that the homozygous wild type genotype (10/- 10) was common in infertile men (77% - 47/61) and in normozoospermic control men (71.7% - 43/60). Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis.

Hepatitis B Viral Surface Mutations in Patients with Adefovir Resistant Chronic Hepatitis B with A181T/V Polymerase Mutations

The hepatitis B virus (HBV) polymerase gene has overlapping reading frames with surface genes, which allows to alter the amino acid codon of the surface genes. In adefovir (ADV) treated chronic hepatitis B patients carrying rtA181T/rtA181V mutations, overlap with surface gene mutations such as sW172stop/sL173F has been reported. However, the clinical consequences of such surface mutations have not been determined. The aim of this study was to determine the surface gene sequence in ADV-resistant patients carrying the A181T/V mutation and to describe the clinical significance. Of the 22 patients included in this study, 13 were ADV-resistant with rtA181T/V mutations (polymerase mutation group, Group P) and nine were antiviral treatment-naive (control group, Group C). The Pre-S1 gene mutation, V60A, was detected in 11 patients (Group P=8, Group C=3). A start codon mutation in the Pre-S2 gene was found in five patients (Group P=3, Group C=2). An S gene mutation, sA184V, was found in nine patients, all of whom were in group P. Although sW172stop and sL173F mutations were detected, reduced HBsAg titer was not observed. Further study of these mutations and their clinical implications are needed.

A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods.

BACKGROUND: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. OBJECTIVES: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. STUDY DESIGN: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol ) gene of HSV isolates. RESULTS: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). CONCLUSION: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.

Translesion DNA polymerases Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa.

Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol zeta, is a highly polymorphic locus in Neurospora.

Overlapping Gene Mutations of Hepatitis B Virus in a Chronic Hepatitis B Patient with Hepatitis B Surface Antigen Loss during Lamivudine Therapy

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yrold Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.

Análisis molecular de la hemofilia A en familias del noroeste de México / molecular analysis of Hemophilia A in northeastern mexican families

Empleando las técnicas de Southern para la detección de genes por hibridación con una sonda radiactiva de la reacción en cadena de la polimerasa para la amplificación de fragmentos génicos, se practicó el análisis molecular a nivel de ADN para detectar portadoras de la hemofilia A. Este se basó en el estudio del polimorfismo para la enzima de restricción Bcl I presente en el intró 18 del gen para el factor VIII de la coagulación. Se analizaron ocho familias de noroeste de México con antecedentes de la enfermedad, que comprendieron un total de 43 individuos. De las 27 mujeres que formaban parte de la muestra, 17 requerían el diagnóstico de portadora, el cual se estableció en tres de ellas, se excluyó en cinco y no se pudo determinar en nueve. Las frecuencias encontradas en la muestra para el polimorfismo fueron de 63 por ciento para el alelo de tamaño y de 37 por ciento para el alelo de tamaño menor, determinando un porcentaje de mujeres heterocigotas (informativas) de 48.2 por ciento y fijando en este valor la utilidad teórica del polimorfismo en las familias aquí incluidas. Estas frecuencias muestran simulitud con las de las poblaciones mediterráneas y establecen que la utilidad del polimorfismo es mayor en nuestra muestra que en otros grupos étnicos.