THE USE OF A SHUTTLE PLASMID TO STUDY NONTARGETED MUTAGENESIS AND ITS SEQUENCE SPECIFICITY

Intact pZ189 DNA was replicated in monkey kidney vero cells which had been pretreated with Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) . The mutants were selected in E. coli MBM7070 and the mutation frequencies involving mutants with unchanged electrophoretic mobilrty of their plasmid DNA were scored. When compared to the spontaneous mutation frequency. the mutation frequencies were increased by 5.8 and 2.9-fold in cells pretreated with 0. 2 and 2μmol/L MNNG, respectively. The supF genes of these mutants were sequenced. and it was found that the types of base substitution and the sites of frameshifts differed from findings in studies of spontaneous and targeted mutagenesis. The results suggest that nontargeted mutagenesis occurs in mammalian cells and may have a sequence specificity.

The application of sequence specific primer and RT-PCR to LRRK2 gene polymorphism typing

Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.

Study on Genomic Changes in Partial Amphiploids of Common Wheat_Wheatgrass

According to conventional theory, little genomic changes should occur in homozygous and stable amphiploids of the grass family, particularly those involving polyploid wheat as a parent. In the present study, however, extensive genomic changes were detected in two octoploid partial amphiploids of common wheat (Triticum aestivum L.)_wheatgrass (Agropyron intermedium (Host) P.B.=Elytrigia intermedia (Host) Nevski=Thinopyrum intermedium (Host) Barkworth and Dewey), namely Zhong 3 and Zhong 5, by RFLP analysis using 10 low_copy, wheat chromosome_specific sequences and 33 representative homoeologous group_specific sequences as probes. Genomic changes involved loss of wheat hybridization fragment(s) and/or acquisition of new fragment(s). Uniformity of the RFLP patterns among 5 individual plants taken respectively from Zhong 3 and Zhong 5 in two successive generations, suggested that genomic changes probably had occurred in the early few generations after octoploid amphiploid formation, and remained essentially static thereafter. The highly similar RFLP patterns between Zhong 3 and Zhong 5, which had identical genomic constitution but differed from each other due to involvement of different wheat varieties as parents imply that genomic changes were probably not at random. Possible causes for the extensive and rapid genomic changes in the newly formed plant amphiploids, as well as their implications for polyploid genome evolution and breeding application are discussed.

The Sequence Modeling Method Based on ECCin Developing Program Specifications

This article discusses the developing process of the version sequencesof specifications and the formal expressions of various reconstructions including theexpansion and revision of the version at each stage. The author suggests using ECC(Extended Calculus of Construction) to describe the specifications of formal systemand using functional language ML to implement this developing process.

Fast infectious diseases diagnostics based on microfuidic biochip system

Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity,specificity,and low consume of sample and reagent is keyword to low cost molecular diagnostics.In this paper,a sensitive DNA isothermal amplifi-cation method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate(PC)microfuidic chip.A portable confocal optical fuo-rescence detector was specifically developed for the microfuidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background.The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65 aM,and a detection limit of less than five copies of genomic DNA.Contrast to the general polymerase chain reaction(PCR)at eppendorf(EP)tube,the detection time in our developed method was reduced from 1.5h to 45 min for multi-target parallel detection,the consume of sample and reagent was dropped from 25μL to 1.45μL.This novel microfuidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets,with potential applications in biotechnology,medicine,and clinical molecular diagnostics.

Analysis of Allele Specific Expression——A Survey

Allele specific expression is essential for cellular programming and development and the diversity of cellular phenotypes. Traditional analysis methods utilize RNA and depend on single nucleotide polymorphisms,thus to suffer from limited amount of materials for analysis. The rapid development of next-generation sequencing technologies provides more comprehensive and powerful approaches to analyze the genomic, epigenetic, and transcriptomic data, and further to detect and measure allele specific expressions. It will potentially enhance the understanding of the allele specific expressions, their complexities, and the effect on biological processes. In this paper, we extensively review the state-of-art enabling technologies and tools to analyze, detect, and measure allele specific expressions, compare their features, and point out the future trend of the methods.

Transcriptome analysis reveals long noncoding RNAs involved in fiber development in cotton (Gossypium arboreum)

Long noncoding RNAs （lncRNAs） play important roles in various biological regulatory processes in yeast, mammals, and plants. However, no systematic identification of lncRNAs has been reported in Gossypium arboreum. In this study, the strand-specific RNA sequencing （ssRNA-seq） of samples from cotton fibers and leaves was performed, and lncRNAs involved in fiber initiation and elongation processes were systematically identified and analyzed. We identified 5,996 lncRNAs, of which 3,510 and 2,486 can be classified as long intergenic noncoding RNAs （lincRNAs） and natural antisense transcripts （IncNAT）, respectively. LincRNAs and lncNATs are similar in many aspects, but have some differences in exon number, exon length, and transcript length. Expression analysis revealed that 51.9% of lincRNAs and 54.5% of lncNATs transcripts were preferentially expressed at one stage of fiber development, and were significantly highly expressed than protein-coding tran- scripts （21.7%）. During the fiber and rapid elongation stages, rapid and dynamic changes in lncRNAs may contribute to fiber development in cotton. This work describes a set of lncRNAs that are involved in fiber development. The characterization and expression analysis of lncRNAs will facilitate future studies on their roles in fiber development in cotton.

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.