Regulable toehold lock for the effective control of strand displacement reaction sequence and circuit leakage

Strand displacement reaction enables the construction of enzyme-free DNA reaction networks,thus has been widely applied to DNA circuit and nanotechnology.It has the characteristics of high efficiency,universality and regulatability.However,the existing regulation tools cannot enable effective control of the reaction sequence,which undoubtedly limits the construction of complex nucleic acid circuits.Herein,we developed a regulation tool,toehold lock,and achieved strict control of reaction sequence without loss of the main reaction signal output.Furthermore,we applied the tool to scenarios such as seesaw circuits,AND/OR logic gates,and entropy-driven circuits,and respectively demonstrated its significant superiority compared to the original method.We believe that the proposed toehold lock has greatly optimized the efficiency of DNA strand displacement-based networks,and we anticipate that the tool will be widely used in multiple fields.

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

In Egypt,protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy.In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected.Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared.The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques.Of the 15 skin nodules suspected of LSD,10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive.An antigenic correlation between field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera.Also,nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared.The results revealed that the four used viruses were antigenically identical.Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain.Thus,further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.

Mossbauer Spectrum Study on Hydrogen Reduction of Preoxidized Ilmenite

Reduction behavior of preoxidized synthetic ilmenite was studied by means of Mossbauer spectra. In the course of reduction, iron component in the samples is reduced in stepwise: Fe 3+ →Fe 2+ →Fe 0, and some intermediate compounds, such as Fe 3O 4, Fe 2TiO 4 etc . and their solid solution may be generated besides Fe 2O 3 and FeTiO 3. If the reduction temperature is higher than 1173 K, the metallization needs only half time of the case when the temperature is lower than 1073 K.

DETECTION OF HUMAN PAPILLOMAVIRUS TYPES 16, 18 DNA RELATED SEQUENCES IN BRONCHOGENIC CARCINOMA BY POLYMERASE CHAIN REACTION

In studying the relationship between human papillomavirus (HPV) and bronchogenic carcinoma, 'high-risk' HPV 16, 18 DNA sequences were detected in samples from 50 lung cancer patients, 18 patients with benign pulmonary diseases and 4 fetal lung tissues by polymerase chain reaction (PCR) and dot-blot hybridization with biotin-labelled probes. The results showed that HPV 16, 18 DNA related sequences were found in 32% of lung cancer specimens, with 10 cases of HPV 16, 5 cases of HPV 18 and 1 case of both types. 48.15% (13 / 27) of squamous cell carcinomas were shown to be positive for HPV 16, 18 DNA. In addition, two adenocarcinomas and one small cell carcinoma were positive for HPV 16 DNA. No specimens from benign diseases tissues and fetal lung tissues showed positive results. These results suggest that primary bronchogenic carcinoma is related to HPV infection.

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N- terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it provides valuable information about the sequence of an unknown peptide. The FAB mass spectra contain a number of characteristic ions at low-mass region in addition to the sequence ions at high-mass region. It was found that the ions below m/z 200 are characteristic of the amino acid composition of the peptide, from which the amino acid composition of the peptide could be estimated. Additionally, mixture analysis is also discussed.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci

Genotyping for Kidd, Kell, Duffy, Scianna, and RHCE blood group antigens polymorphisms in Jiangsu Chinese Han

Background Molecular testing is more precise compared to serology and has been widely used in genotyping blood group antigens. Single nucleotide polymorphisms （SNPs） of blood group antigens can be determined by the polymerase chain reaction with sequence specific priming （PCR-SSP） assay. Commercial high-throughput platforms can be expensive and are not approved in China. The genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE blood group antigens in Jiangsu province were unknown. The aim of this study is sought to detect the genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE antigens in Jiangsu Chinese Hart using molecular methods with laboratory developed tests. Methods DNA was extracted from EDTA-anticoagulated blood samples of 146 voluntary blood donors collected randomly within one month. Standard serologic assay for red blood cell antigens were also performed except the Scianna blood group antigens. PCR-SSP was designed to work under one PCR program to identify the following SNPs： JK1/JK2, KEL 1/KEL2, FYA/FYB, SC1/SC2, C/c and E/e. Results Serologic antigen results were identical to the phenotypes that were predicted from genotyping results. The allele frequencies for Jk^＊01 and Jk^＊02 were 0.51 and 0.49, respectively; for Fy^＊A and Fy^＊B 0.94 and 0.06; for RHCE^＊C and RHCE^＊c 0.68 and 0.32; and for RHCE^＊E and RHCE^＊e 0.28 and 0.72. Among 146 blood donors, all were KEL^＊02/ KEL^＊02 and SC^＊01/SC^＊01, indicating allele frequencies for KEL^＊02 and SC^＊01 close to 1.00. Conclusions The use of PCR-SSP working under the same condition for testing multiple antigens at the same time is practical. This approach can be effective and cost-efficient for small-scale laboratories and in developing counties. These molecular tests can be also used for identifying rare blood types.

ISSR markers based on GA and AG repeats reveal genetic relationship among rice varieties tolerant to drought,flood,or salinity

Drought, flood, salinity, or a combination of these limits rice production. Several rice varieties are well known for their tolerance to specific abiotic stresses. We determined genetic relationship among 12 rice varieties including 9 tolerant to drought, flood, or salinity using inter-simple sequence repeat （ISSR） markers. Based on all markers, the nine tolerant varieties formed one cluster distinct from the cluster of three control varieties. The salt-tolerant varieties were closest to two flood-tolerant varieties, and together they were distinct from the drought-tolerant varieties. （GA）8YG was the most informative primer, showing the highest polymorphic information content （PIC） and resolving power （Rp）. The drought-, flood-, and salt-tolerant varieties grouped in three distinct clusters within the group of tolerant varieties, when （GA）8YG was used. Sabita was the only exception. The two aus varieties, Nagina22 and FR13A, were separated and grouped with the drought- and flood-tolerant varieties, respectively, hut they were together in dendrograms based on other primers. The results show that ISSR markers associated with （GA）sYG delineated the three groups of stress-tolerant varieties from each other and can be used to identify genes/new alleles associated with the three abiotic stresses in rice germplasm.

Study on vertical transmision of Chlamydia trachomatis using PCR and DNA sequencing

Objective To investigate the vertical transmission rate of Chlamy dia trachomatis (CT) in Chongqing, China. Methods Specimens taken from 278 women and from their 79 infants were e xamined b y cell culture, polymerase chain reaction (PCR) and DNA sequence analysis. Chlam ydia trachomatis was isolated in McCoy cell culture. CT DNA was extracted with a modified NaI method. After cloning, recombinant plasmids were used for sequence analysis with the dideoxy chain termination method.Results 10.8% (30/278) of the cervical cultures of pregnant women were positive for Chlamydia trachomatis, while the positive rate tested by PCR was 14.0% (39/2 78). The vertical transmission rate of Chlamydia trachomatis was 55. 0% (11/20). The incidences of conjunctivitis and pneumonia in infants with Chlamydia t rachom atis positive mothers were 27.3% and 18.2%, respectively. DNA sequence s of Chlam ydia trachomatis isolated from the cervix of a mother and the nasopharynx of her baby were identical.Conclusion Chlamydia trachomatis infection is quite common in Cho ngqing , China. Our report is the first report of CT vertical transmission proved by DN A sequence analysis.