Genome, Human ; genetics ; Humans ; Models, Theoretical ; Neoplasms ; genetics ; Sequence Analysis, DNA ; methods

BACKGROUND AND OBJECTIVEGlutathione S-transferase M1 (GSTM1) is an important phase II metabolic enzyme gene which involves metabolism of various carcinogens in human body. Many studies showed that GSTM1 genetic polymorphism was associated with lung cancer risk. The aim of this study is to investigate the relationship between GSTM1 genetic polymorphism and lung cancer risk among Han nationality population in Tianjin district.

METHODSGSTM1 genetic polymorphism was detected by melting curve analysis of SYBR green I real-time PCR assay. Two hundred and sixty-five histological confirmed lung cancer patients and 307 health controls were recruited in this case-control study and the relationship between GSTM1 genetic polymorphism and lung cancer riskwas investigated.

RESULTS(1) The frequency of the GSTMI(-) in lung cancer and control groups was 56.6% and 57.0% respectively, and no significant difference was found between the distribution of the GSTM1 (-) genotype in the two groups (chi2 = 0.831, P = 0.362). (2) When considered the GSTM1(+) genotype as reference, there was no overall statistically increased lung cancer risk for carriers with the GSTM1(-) genotype adjusted by age, gender and smoking status (OR = 0.840, 95% CI: 0.578-1.221, P = 0.362). (3) The frequency of the GSTM1(-) genotype for squamous cell carcinoma, adenocarcinoma, SCLC and other histological types was 65.8%, 48.5%, 47.8% and 52.2% respectively, compared with the control group, no statistically increased lung cancer risk was observed (P > 0.05).

CONCLUSIONNo evidence is found between GSTMI genetic polymorphism and lung cancer risk among Han nationality population in Tianjin district.

Adult ; Aged ; Female ; Genotype ; Glutathione Transferase ; genetics ; Humans ; Lung Neoplasms ; etiology ; genetics ; Male ; Middle Aged ; Organic Chemicals ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Risk

BACKGROUND AND OBJECTIVEIt has been proven that cytochrome P450 enzyme 2A13 (CYP2A13) played an important role in the association between single nucleotide polymorphisms (SNP) and human diseases. Cytochrome P450 enzymes are a group of isoenzymes, whose sequence homology may interfere with the study for SNP. The aim of this study is to explore the interference on the SNP study of CYP2A13 caused by homologous sequences.

METHODSTaqman probe was applied to detect distribution of rs8192789 sites in 573 subjects, and BLAST method was used to analyze the amplified sequences. Partial sequences of CYP2A13 were emplified by PCR from 60 cases. The emplified sequences were TA cloned and sequenced.

RESULTSFor rs8192789 loci in 573 cases, only 3 cases were TT, while the rest were CT heterozygotes, which was caused by homologous sequences. There are a large number of overlapping peaks in identical sequences of 60 cases, and the SNP of 101 amino acid site reported in the SNP database is not found. The cloned sequences are 247 bp, 235 bp fragments.

CONCLUSIONThe homologous sequences may interfere the study for SNP of CYP2A13, and some SNP may not exist.

Aryl Hydrocarbon Hydroxylases ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics

Background and objective hTe methods for introducing point mutations into target genes are impor-tant for dissecting the relationship of gene structure and function. Up to date, there are numbers of protocols available for the purpose. However, many of them are suited for introducing single site mutation into the target gene. For introducing multiple-site mutations simultaneously into the target genes, it is required to further improve the related methods. Methods In this report, we describe an improvement on the type IIs restriction enzyme-dependent site-directed mutagenesis method and the improved protocol is highly effcient for multiple-site mutagenesis. In our method, a pair of mutagenic primers are synthesized for each desired site and each mutagenic primer contains a selected type IIs restriction site. hTe DNA fragments between two neighboring sites are ampliifed with PCR. All ampliifed fragments are then digested by the selected Type IIs restriction enzyme. hTe expected mutant is eventually generated by ligation of these digested DNA fragments. Results hTe improved protocol is very easy and can be achieved in just one day. As a proof of principle, we have introduced multiple-site, i.e., 3-site or 4-site mu-tations into the fusion gene of nm23 and EGFP (enhanced green lfuorecence protein). hTe mutagenic frequencies are almost reached 100%. Conclusion Our protocol provides a useful tool for gene function research.

Background and objective In vitro site-directed mutagenesis is a routine technique in molecular biol-ogy labs. However, although there are numbers of related methods available, most of these methods are not suitable for intro-ducing mutations into large vectors. Methods In this report, we describe a method which is highly effective for this purpose. Our method is based on the other site-directed method we recently reported. hTe basic protocol of our method is as follows:(1) Synthesize a pair of vector primers based on the sequences around the region to be mutated, each containing a suitable type IIs endonuclease restriction site;meanwhile, synthesize a pair of short complementary oligonucleotides which forms a mutagenic fragment atfer annealing;(2) Synthesize a pair of bridge primers which can speciifcally bind to a site in the vector sequence, each containing a suitable type IIs endonuclease restriction site;(3) Perform PCR reactions using these Vector primers and Bridge primers;(4) Digest the PCR products with the corresponding type IIs restriction enzyme;(5) Ligate the digested frag-ment with the mutagenic fragment to make the desired mutant. Results Using this protocol, we have introduced mutations into a vector larger than 9 kb. hTe results shows that the mutation rates are more that 90%. Conclusion Our method provides a useful tool for performing site-directed mutagenesis experiment in large vector.

Lentiviral vectors are currently very effective tools in molecular and cell experiment. Lentiviral vector, a kind of retroviral vectors, has a number of unique advantages in target gene transferation, for example, the ability of transfection to the dividing or nondividing cells, its high effciency of transfection and a capacity of large target gene fragments. hTis paper describes the sources of lentiviral vectors, molecular characteristics, research progress,etc.

Octamer-binding transcription factor 4 (OCT4) belongs to the POU-homeodomain family of tran-scription factors and binds to an octamer motif, ATGCAAAT. OCT4 is the key transcription factor that is involved in the maintenance of pluripotency and self-renewal in undiferentiated embryonic stem (ES) cells. OCT4 has been reported to be overexpressed in various cancers including lung, germ cell tumors, breast, cervix, prostate, gastric, liver, and ovarian cancer. Mi-croRNAs (miRNAs), small non-coding RNAs, modulate mRNA expression through base pairing between seed sequences in miRNA and complementary sequences of the target mRNA, thereby destabilizing mRNA and/or inhibiting protein synthesis. Several miRNAs have been demonstrated to regulate stemness factors such as OCT4, NANOG, SOX2 and KLF4 in cancer cells, thereby modulating the proliferation, apoptosis, diferentiation, drug resistance and immunity of cancer cells.