OBJECTIVETo establish a novel method for the multiplex analysis of the methylation and single nucleotide polymorphism (SNP).

METHODSThe imprinted SNP rs220028 was chosen as a model. Genomic DNA, after being digested with methylation sensitive restriction enzyme, were typed by mutagenically separated PCR (MS-PCR). The polymorphism of restriction site was excluded by PCR-RFLP.

RESULTSBy post-digestion MS-PCR, the methylated allele was detected selectively, the maternal origin of which was confirmed by pedigree analysis; A=0.5085, G=0.4915,PIC=0.3749.

CONCLUSIONThe multiplex analysis of methylation markers and SNP can be achieved by post-digestion MS-PCR. The imprinted SNP locus rs220028 is a potentially useful marker in screening Prader-Willi/Angelman syndrome.

DNA Methylation ; DNA Restriction Enzymes ; metabolism ; Genetic Markers ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; Polymorphism, Single Nucleotide

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Restriction Enzymes ; metabolism ; DNA, Neoplasm ; genetics ; Glutathione S-Transferase pi ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods

Differences in ability to produce the specific phenolic glycolipid-Tb (PGL-Tb) antigen among Mycobacterium tuberculosis strains have been reported. One of the explanations would be the genotypic variation between the strains. In this study, we compared the DNA fragment patterns after digestion of DNA with various restriction enzymes between the PGL-Tb producing and non-producing strains of M. tuberculosis. Three clinical isolates of M. tuberculosis producing the PGL-Tb antigen detectable by thin-layer chromatography, and M. tuberculosis H37Rv and M. bovis BCG not producing the antigen were grown in Sauton medium. The chromosomal DNA was digested with the restriction endonucleases, Eco RI, Sau3A I, BamH I, Xho I, Sma I, Pst I, Hinc II, and Bst EII. Most of the restriction enzymes used gave no clear DNA bands or no DNA fragment common just to the PGL-Tb producing strains. When DNAs were digested with Bst EII, however, there was a 13 kb DNA fragment common to the PGL-Tb producing isolates of M. tuberculosis and not present in the H37Rv strain and M. bovis BCG. This study thus suggests that there might be differences in DNA fragment patterns between the PLG-Tb producing and non-producing strains of M. tuberculosis.
Base Sequence ; Comparative Study ; DNA Restriction Enzymes ; DNA, Bacterial/*metabolism ; Glycolipids/*biosynthesis ; Molecular Sequence Data ; Mycobacterium tuberculosis/*genetics/metabolism ; Support, Non-U.S. Gov't ; Tuberculosis/microbiology

OBJECTIVETo search for the genes which could interact with newly found homo sapiens cross-immune reaction antigen (PCIA1) gene and accordingly to provide insights into the study of the gene function.

METHODSThe Stratagene's BacterioMatch Two-Hybrid System and BacterioMatch Fetal Kidney Library were adopted and the recombinant bait plasmid pBT-PCIA1 was cotransformated with the target plasmid pTRG-cDNA library DNA into the reporter stain. After screening and isolation of positive pTRG clones, the target genes were identified by DNA sequencing and bioinformation analysis.

RESULTSAmong all the seven detected target genes, three genes' function were not known, the other four genes had important functions. Their mutations or abberant expression resulted in severe diseases and overexpression of ACTN4 (actinin, alpha 4), PSAP (prosaposin) or EIF3S10 (eukaryotic translation initiation factor 3, subunit 10 theta) could promote tumor development and progression.

CONCLUSIONThe bacterial two-hybrid system technique is an efficient method, which can provides insights into the study of novel genes' function by detecting protein-protein interactions. This study indicates that PCIA1 gene expression correlates with tumor formation, invasion and metastasis.

Bacteria ; genetics ; metabolism ; Computational Biology ; DNA Restriction Enzymes ; metabolism ; Gene Library ; Genetic Vectors ; Humans ; Neoplasms ; genetics ; pathology ; Plasmids ; genetics ; Sequence Analysis, DNA ; Two-Hybrid System Techniques

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 101 to 102 oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.
Cryptosporidium/*isolation & purification ; Cyclospora/*isolation & purification ; DNA Primers/genetics ; DNA Restriction Enzymes/metabolism ; DNA, Protozoan/genetics/metabolism ; Humans ; Microsporidia/*isolation & purification ; Parasitology/*methods ; Polymerase Chain Reaction/*methods ; Polymorphism, Restriction Fragment Length ; Sensitivity and Specificity ; Water/*parasitology

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.
Atractylodes ; classification ; genetics ; DNA Restriction Enzymes ; metabolism ; DNA, Plant ; genetics ; Drug Contamination ; Genotype ; Lonicera ; classification ; genetics ; Plants, Medicinal ; classification ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.

METHODSIP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.

RESULTSPCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.

CONCLUSIONA eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.

Animals ; Chemokine CXCL10 ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; Fluorescent Antibody Technique ; Gene Expression ; Genetic Vectors ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Rats ; Transfection ; methods

OBJECTIVETo determine whether vitamin D receptor(VDR) gene start codon polymorphisms and 3'-end region polymorphisms exerted a combined influence on bone mineral density(BMD) in Han postmenopausal women in Beijing area.

METHODSThe VDR Fok I and 3'-end region genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 110 unrelated postmenopausal women. BMD was measured at the lumbar spine (L(2-4)), femoral neck(Neck), Ward's triangle(Ward's) and trochanter (Troch) using duel-energy X-ray absorptiometry.

RESULTSThe frequencies distribution of Fok I, Apa I, Bsm I and Taq I alleles in this cohort all followed the Hardy-Weinberg equilibrium. No significant association of Fok I, Apa I or Taq I genotype with BMD in postmenopausal women was found when these polymorphisms were considered independently, except for Bsm I genotype. When a combined analysis of VDR gene Fok I and 3'-end region polymorphisms was carried out, cross-genotyping Fok I and Apa I polymorphisms was significantly associated with BMD at the L(2-4) (P<0.001), and cross-genotype of Fok I and Taq I was also significantly associated with BMD at the Neck and Troch sites (P<0.05). However, cross-genotyping Fok I and Bsm I polymorphisms was not significantly associated with BMD. Cross-genotyping Apa I and Bsm I or Taq I polymorphisms was not associated with BMD in postmenopausal women, either.

CONCLUSIONAlthough Fok I polymorphisms of VDR gene were not significantly associated with BMD in postmenopausal women, VDR gene Fok I and 3'-region polymorphisms (Apa I and Taq I) had a combined effect on the BMD in postmenopausal women.

3' Flanking Region ; genetics ; Aged ; Analysis of Variance ; Bone Density ; physiology ; China ; Codon, Initiator ; genetics ; DNA ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; Female ; Gene Frequency ; Genotype ; Humans ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Postmenopause ; genetics ; physiology ; Receptors, Calcitriol ; genetics

In order to prevent phage contamination in amino acid fermentation, we introduced the restriction and modification system cglI gene complex into Corynebacterium crenatum and studied their phage-resistance. The cglI gene complex was amplified from Corynebacterium glutamicum by PCR and constructed into pJL23 vector. The recombinant strains were obtained by transformation of the recombinant plasmid pJL23-cglI into C. crenatum. Results showed that the recombinant strains possessed strong phage-resistance activity and broad phage-resistance spectrum, demonstrating the feasibility of using cglI gene complex for construction of phage-resistance recombinant C. crenatum strains and presenting a powerful way to solve the problem of phage contamination in amino acid fermentation industry.
Amino Acids ; biosynthesis ; Bacterial Proteins ; genetics ; Bacteriophages ; growth & development ; Corynebacterium ; genetics ; virology ; Corynebacterium glutamicum ; genetics ; metabolism ; DNA Restriction-Modification Enzymes ; genetics ; Fermentation ; Galectins ; genetics ; Recombination, Genetic ; Transformation, Bacterial

Familial benign hypocalciuric hypercalcemia (FBHH) is an autosomal dominant trait with high penetrance, clinically manifestating a relatively benign, lifelong, persistent hypercalcemia and hypocalciuria without hypercalcemic related complications. The calcium-sensing receptor (CaSR) plays an important role in the regulation of PTH secretion and calcium metabolism. Here we present a family with FBHH of an autosomal dominant inheritance. A heterozygous mutation of E297K (GAG -> AAG, exon 4) of CaSR gene was found in 3 family members. To our knowledge, it is the first confirmed case of FBHH with CaSR gene mutation in Korea.
Sequence Analysis, DNA ; Receptors, Calcium-Sensing/*genetics ; Pedigree ; Parathyroid Hormone/analogs & derivatives/genetics/metabolism ; *Mutation ; Metabolism, Inborn Errors/*genetics ; Male ; Korea ; Hypercalcemia/*genetics ; Humans ; Heterozygote ; Genes, Dominant ; Female ; Family Health ; Exons ; DNA Restriction Enzymes/metabolism ; DNA/metabolism ; Adult