Biomedical subjects

Guo-hua Zhou

Publications and source records attributed to Guo-hua Zhou.

4 recordsLinked to original sources

PubMed2006

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification.

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.

Alleles↗

PubMed2003

[Characterization of N-glycan mapping of bioengineering recombinant erythropoietin by capillary electrophoresis with laser-induced fluorescence].

AIM: N-Glycans in recombinant human erythropoietin (EPO) are essential to in vivo biological activity. This paper is to develop a method for mapping sialyated or asialyated N-glycans of EPO. METHODS: At first, N-glycans linked to asparagines in glycoprotein EPO were released by peptide N-glycosidase F. To map asialyated N-glycans, sialic acid in N-glycans were removed by incubating N-glycans with sialidase. Oligosaccharides were labeled with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate (APTS), and all of the labeled oligosaccharides released from EPO were mapped by capillary gel electrophoresis with laser-induced fluorescence. The relationship between N-glycans and bioactivity of EPO was investigated on the basis of N-glycan mapping spectra. RESULTS: N-Glycans of seven different batches of EPO were mapped. Each sample was analysed twice, with and without sialidase treatment. The results showed that N-glycans of each sample were approximately the same. But when the expression vector was different, the types of N-glycans and their relative content were quite different. In case of asialyated N-glycan mapping, the retention time of each oligosaccharide delayed greatly, and most importantly, the resulted sialic acid peak can be used as a quantitative standard to determine sialic acid content in N-glycans of EPO. In addition, the difference of N-glycan mapping was observed when the in vivo biological activity of EPO was different. CONCLUSION: The approach in this article for determining N-glycan mapping can be applied to determine the source of EPO and the difference between each batch. It is also a suitable tool for routinely controlling the inner quality of EPO by coupled with peptide mapping.

Electrophoresis, Capillary↗

PubMed2002

[Active immunity for anti-colorectal cancer induced by chemokine MCP-3 gene transfection].

BACKGROUND & OBJECTIVE: Chemokines play an important role in the infiltration of immune cells to tumor tissues. Anti-tumor immune response had been elicited in many tumor models by the chemokine gene transfection. The aim of this study was to evaluate the possibility of inducing anti-colorectal cancer active immune response by transfection of mouse colorectal cancer CMT93 cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by chemotaxis assay. In vivo experiments were performed to observe the tumorigenicity of wild type CMT93 and MCP-3 gene modified tumor cells. The immune cell infiltration in tumor tissues and tumor metastasis were detected histopathologically. RESULTS: RT-PCR detection showed MCP-3 was expressed in MCP-3 gene-transfected G418-resistant clones(CMT93/MCP-3), but not in wild type CMT93. In chemotaxis assay, the results showed that the cell culture supernatant of CMT93/MCP-3 possess obviously chemotactic activity. The chemotactic index of the CMT93/MCP-3 supernatant was 5.57(P < 0.05). The supernatants from the control groups did not possessed the chemotactic activity. In vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased significantly compared to wild type CMT93, but the tumors grew more slowly from CMT93/MCP-3 than from the controls (P < 0.05). In the tumor tissue from CMT93/MCP-3, obvious infiltrated immune cells were found, and few immune cells infiltrated in the tumor tissue from the controls. In the mice inoculated with CMT93/MCP3 tumor cells, tumor metastasis was inhibited significantly, its metastasis rate was 0(0/7), lower than that of CMT93 (100%, 4/4) and CMT93/mock (80%, 4/5) (P < 0.05). CONCLUSION: Transfection with chemokine MCP-3 gene can induce anti-colorectal cancer active immune response, but the tumor growth cannot be inhibited completely by merely MCP-3 gene transfection.

Animals↗

PubMed2002

[P53 gene mutation detection by bioluminometry assay].

AIM: To develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation. METHODS: Pyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step-by-step using four enzymes, DNA-polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single-stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence. RESULTS: A new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0.2 microL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant. CONCLUSION: The developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.

Exons↗