Zinc finger nuclease (ZFN), which is a chimeric fusion structure between a Cys2-His2 zinc-finger protein (ZFP) and the cleavage domain of Fok I endonuclease, can be used to introduce targeted double-stranded breaks (DSBs). ZFN-mediated cleavage leads to mutations when double-stranded breaks are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). In recent years, ZFNs are widely used in the fields of genetic research. In this review, the methodology and technical advantages of ZFNs were briefly discussed.
Animals ; Deoxyribonucleases, Type II Site-Specific ; chemistry ; genetics ; metabolism ; Humans ; Zinc Fingers

OBJECTIVETo construct a reverse transcriptase-polymerase chain reaction (RT-PCR)approach that can improve the specificity of primers while dropping down the nonspecific amplification.

METHODSIn the recent study we reported a new RT-PCR assay which improved markedly the specificity. However its efficiency of regressing nonspecific amplification remains to be accurately checked and further documented. In primer design, we looked over again some sequences that showed differences at 5' or 3' ends between human CAV1 and mouse Cav1 genes. cDNAs and the diluted plasmids which harbored the sequence of human CAV1 or mouse Cav1 gene were chosen as the templates. The ordinary PCR compared with one, of which primers modified by phosphorothioate and combined with proofreading polymerase, for their efficiencies of nonspecific amplification inhibited.

RESULTSTaq DNA polymerase without proofreading activity could efficiently catalyze the extension of primers with a single or multiple mismatched base pairs at the 3' terminus, but the kind of primer extension can be effectively blocked by phosphorothioate modified primers combined with proofreading polymerase. Compared with ordinary PCR reaction, this new PCR method can effectively regress the primer mismatched amplification of 50 ng DNA almost equaling to 2 x 10(4) unmatched template copies in a final volume of 50 microL.

CONCLUSIONCompared with the first generation of polymerases with or without proofreading activities mediating RT-PCR reaction, the introduction of nuclease-resistant 3' modified primers (3' phosphorothioate primer extension) can offer more simplicity, accuracy, and also decrease cost.

Animals ; Caveolin 1 ; genetics ; Deoxyribonucleases ; metabolism ; Gene Amplification ; Humans ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Thionucleotides ; metabolism

UNLABELLEDTo identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp.

CONCLUSIONSSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

Cloning, Molecular ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Fetus ; Gene Library ; Humans ; Mesoderm ; cytology ; metabolism ; Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism

The purpose of this study is to investigate the Sal I, Nru I and Mse I restriction fragment length polymorphisms (RFLPs) of factor IX gene in Chinese Han people. The frequencies of FIX-192 and FIX-793 for A and G, and FIX-698 for T and C were analyzed by polymerase chain reaction (PCR) in unrelated normal Chinese Han people. A sample of 214, 210 and 206 unrelated X chromosomes were analyzed for FIX-192 and FIX-793 and FIX-698, respectively. The results showed that the frequencies for FIX-192 were 0.878 for A and 0.122 for G, with a heterozygosity rate of 0.213, and the frequencies for FIX-793 were 0.552 for A and 0.448 for G, with a heterozygosity rate of 0.494, the frequencies for FIX-698 were 0.311 for T and 0.689 for C, with a heterozygosity rate of 0.429. It was concluded that the SalIand NruI and MseI RFLPs of FIX gene may be useful markers for carrier detection and prenatal diagnosis in Chinese families with hemophilia B patients.
China ; DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Factor IX ; genetics ; Female ; Gene Frequency ; Heterozygote ; Humans ; Male ; Polymorphism, Restriction Fragment Length

OBJECTIVETo determine the relationship between vitamin D receptor (VDR) gene polymorphism and osteoporosis in postmenopausal women.

METHODSThe polymerase chain reaction-restriction fragment length polymorphism was used to detect VDR genotype in 40 patients with osteoporosis and 21 healthy postmenopausal women.

RESULTSIn the patients with osteoporosis, the bb, Bb, and BB genotype accounted for 82.5%, 17.5% and 0, respectively; in healthy groups, they were 85.71%, 14.29% and 0, respectively (P>0.05).

CONCLUSIONSignificant association between VDR genotype and osteoporosis in Chinese women was observed in this study.

DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Frequency ; Genotype ; Humans ; Middle Aged ; Osteoporosis, Postmenopausal ; genetics ; Polymorphism, Genetic ; Receptors, Calcitriol ; genetics

Adrenal Hyperplasia, Congenital ; genetics ; Chromatography, High Pressure Liquid ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Humans ; Point Mutation ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo establish a rapid method of detecting CYP21 gene mutations.

METHODSFifty Chinese patients with 21-hydroxylase deficiency and some of their families were investigated. Blood samples were obtained for extraction of peripheral blood lymphocytes. A search for restriction sites discriminating between the morbid and the normal in CYP21 gene was made by the computer program DNAssist. PCR-based amplication-created restriction site(PCR-ACRS) was performed at I172N and R356W which are not natural recognition sequence. In addition, I172N and R356W were analysed in five families which conform to the applicability of PCR-ACRS.

RESULTSIn 50 identified 21-hydroxylase deficient Chinese patients, 21 were found to have I172 N (3 were homozygote, 18 were heterozygote); 8 were found to have R356W, all of them were heterozygote. By analysing the families, the findings were consistent with the characteristics of autosomal recessive genetic deficiency.

CONCLUSIONAnalysis of CYP21 gene point mutations using PCR-ACRS is relatively simple, accurate and feasible.

Adrenal Hyperplasia, Congenital ; enzymology ; genetics ; China ; DNA ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Humans ; Male ; Mutation ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Steroid 21-Hydroxylase ; genetics ; metabolism

OBJECTIVETo study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in colorectal cancer and to provide a clue for the mechanism of carcinogenesis of colorectal cancer.

METHODSThe expression levels of IGF2 in the paired colorectal cancer and adjacent normal tissue were examined and compared by use of semi-quantitative reverse transcription-polymerase chain reaction. The imprinting status of IGF2 was detected by restriction fragment length polymorphism. The relationships between the expression level of IGF2, its imprinting status, and the carcinogenesis of colorectal cancer were analyzed.

RESULTSIGF2 was overexpressed in 82.4% (28/34) of colorectal cancer tissues which was significantly higher than those of the matched normal tissues (P<0.01, t=3.01). 87.5% (14/16) of colorectal cancer showed loss of imprinting(LOI), while 71.4%(10/14) of normal tissues also displayed LOI of IGF2.

CONCLUSIONOverexpression of IGF2 was found to play an important role in carcinogenesis of colorectal cancer. LOI of IGF2 may be a prophase manifestation of colorectal cancer.

Colorectal Neoplasms ; genetics ; pathology ; DNA, Neoplasm ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Genomic Imprinting ; Humans ; Insulin-Like Growth Factor II ; genetics ; Male ; Middle Aged ; RNA, Neoplasm ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the genomic variation of Epstein-Barr virus (EBV) and its significance in nasopharyngeal carcinogenesis.

METHODSForty nasopharyngeal carcinoma (NPC) biopsy tissues were used for detection of EBV BamHI f variant and LMP1 XhoI-loss by polymerase chain reaction (PCR), nested PCR, and RFLP (restriction fragment length polymorphism). Forty-eight samples of peripheral blood mononuclear cells (PBMC) taken from apparently healthy adult individuals were used for detection of LMP1 XhoI-loss. Three samples of amplified LMP1 exon 1 DNA from B95-8 cell line and 2 NPC tissues (one having XhoI-loss and the other having Wt-XhoI/XhoI-loss) were sequenced.

RESULTSThirty out of the 40 NPC cases (30/40, 75%) harbored EBV BamHI f variant and the remaining 10 (10/40, 25%) harbored BamHI F prototype. Thirty out of the 39 NPCs (30/39, 76.9%) showed single EBV LMP1 XhoI-loss, 7 (7/39, 18.0%) showed single LMP1 Wt-XhoI (presence of a XhoI site in exon 1 of LMP1 gene, as in B95-8 cell line), and 2 (2/39, 5.1%) showed both LMP1 Wt-XhoI and XhoI-loss. Thirty-eight of the 39 NPCs (97.4%) showed EBV LMP1 XhoI-loss or/and BamHI F variant. In the NPC tissue (1 case only) showing the prototype of Wt-XhoI/BamHI "f", there were several base substitutions, including 5 missense mutations and 2 silent mutations present in LMP1 exon 3, on DNA sequencing. On the other hand, 10 out of the 48 samples of PBMC taken from apparently healthy individuals could be amplified successfully by nested PCR for detection of LMP1 XhoI site. All of these 10 samples carried the prototype of EBV LMP1 Wt-XhoI.

CONCLUSIONSThe majority of EBV present in neoplastic cells of NPC is of BamHI "f" variant and/or possesses LMP1 XhoI-loss, as compared with that in healthy individuals. This genomic variation of EBV may bear some roles in the development and progression of NPC.

Adult ; Aged ; Binding Sites ; genetics ; DNA, Viral ; genetics ; metabolism ; Deoxyribonuclease BamHI ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Mutation ; Nasopharyngeal Neoplasms ; virology ; Sequence Deletion ; Viral Matrix Proteins ; genetics