RESUMEN
CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.
RESUMEN
Objective A very high prevalence of rheumatoid arthritis (RA) is observed in Minnan population in China.We aimed to explore the genetic characteristics of RA in Minnan population and genetic mechanisms of RA by studying the associations of three single nucleotide polymorphisms (SNPs) of signal transducer and activator of transcription 4 (STAT-4) (rs7574865),the cytotoxic T-lymphocyte antigen-4 (CTLA)-4 (rs3087243) and chromosome 9p21.3(rs1333049) with RA in Minnan population.Methods A case-control study of 119 RA patients and 125 normal controls from Quanzhou were enrolled.SNPs (rs7574865,rs3087243,rs1333049) were genotyped by allele-specific polymerase chain reaction (PCR) and analyzed by SPSS 18.0.x2-test was applied to compare allele and genotype frequeneies betweeen cases and controlsLogistic regression models were used to analyze the SNPs.Results The results showed the genotype distributions of STAT4 genes were significantly different between case and control groups (P＜0.01).Compared with the GT heterozygous genotype,TT and GG homozygosity carriers had a lower risk (OR=0498 and 0.300,P=0.018 and P=0.002 respectively).There was not statistical difference in genotypes and allele in CTLA-4 (rs3087243) between RA patients and healthy controls (x2=4.083,P=0.130),but compared with the AG genotyoe,GG homozygosity carriers had a lower risk on basis of statistics (OR=0.580,P=0.04).There was not statistical difference in genotypes and allele in the chromosome 9p21.3 (rs1333049) (P＞0.05),but compared with the GG genotype carriers,CC and GC genotypes carriers had a lower risk on basis of statistics (OR=0.565,P=0.0495).Conclusion Chromosome 9p21.3 (rs1333049) and CTLA-4 rs3087243 G/A may not be associated with susceptibility to RA in Minnan popula-tions.This replication study confirmes that STAT4 rs7574865 G/T polymorphism is associated with susceptibility to RA in Minnan population.
RESUMEN
Objective To improve the tri-primer allele gene amplification for realizing the single nucleotide polymorphisms (SNP) genotyping of the peripheral blood sample .Methods Aiming at the peripheral blood samples with the clinical usual antico-agulation processing by EDTA ,heparin and citrate ,with the locus rs1165205 as the target site ,the buffer solution(YW) suitable for whole blood was prepared and the PCR amplification system and the amplification condition were optimized for realizing the detec-tion of SNP genotyping .Results The genotyping results of locus rs1165205 by improved tri-primer allele gene amplification method were consistent with the results of the Sanger sequencing method ,and the peripheral blood samples treated by different anticoagu-lant were genotyped by the improved tri-primer ASA .Among 80 samples ,various genotypes of locus rs1165205 had no statistical differences in the distribution between the gout population and non-gout population(P= 0 .335) .Conclusion The improved tri-primer allele gene amplification method can be adopted to conduct the rapid genotyping research on gout SNP locus of the peripheral blood samples with the clinical usual anticoagulation processing .
RESUMEN
Objective To explore the association between SLC2A9,SLC17A3,ABCG2 single nucleotide polymorphisms and gout susceptibility in Quanzhou.Methods One hundred and fifty-four cases of gout patients and 160 healthy controls were selected,single nucleotide polymorphisms (SNP) of SLC2A9 SLC17A3,ABCG2 with tri-primer polymerase chain reaction (PCR) were tested and the relation between different genotypes and primary gout prevalence were analyzed.Results High risk genotype frequency of rs16890979 was 93.5％ and 70.0％ in patients and healthy people,respectively (the difference of genotype frequency between the two groups was statistically significant (x2=55.377,P＜0.01).High risk allele frequency was 79.9％ and 48.4％ in patients and healthy people,respectively (allele frequency in different population was statistically significant,x2=67.128,P＜0.01).High risk genotype frequency of rs2231142 was 68.8％ and 38.7％ in patients and healthy people,respectively (the difference of the genotype frequency was statistically significant,x2=29.129,P＜0.01);High risk allele frequency was 43.5％ and 23.4％ in patients and healthy people,respectively (the difference of allele frequency was statistically significant,x2=28.468,P＜0.01) ; rs1165205was a protective SNP,low risk genotype frequency was 42.2％ and 45.6％ in patients and healthy people,respectively (the difference of genotype frequency was statistically significant,x2=0.373,P=0.571); High risk allele frequency was 26.0％ and 28.1％ in patients and healthy people,respectively (the difference of allele frequency was not statistically significant,x2=0.270,P=0.364).Conclusion SNP loci rs16890979 of SLC2A9 gene and rs2231142 of ABCG2 gene can be used as genetic markers for primary gout susceptibility in the Quanzhou area,but SNP loci rs1165205 of SLC17A3 gene has little correlation with the prevalence of primary gout in Quanzhou residents.
RESUMEN
Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.
RESUMEN
Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.