Biomedical subjects

Quanjun Liu

Publications and source records attributed to Quanjun Liu.

8 recordsLinked to original sources

PubMed2006

Microarray-in-a-tube for detection of multiple viruses.

BACKGROUND: The detection of multiple viruses is important for pathogenic diagnosis and disease control. Microarray detection is a good method, but requires complex procedures for multiple virus detection. METHODS: We developed a novel PCR assay, the microarray-in-a-tube system, which integrates multiple PCR processes and DNA microarrays for multiple virus detection. A 5 x 5 oligonucleotide microarray for detecting 4 respiratory tract viruses (severe acute respiratory syndrome-associated coronavirus, influenza A virus, influenza B virus, and enterovirus) with inner controls was arranged on the inner surface of a specially designed Eppendorf cap with a flat, optically transparent window. RESULTS: We were able to perform all detection processes in the encapsulated system without opening the cap. The 4 viruses were successfully amplified by one-step reverse transcription-PCR in the encapsulated tube. After the PCR process, the microarray-in-a-tube was inverted, and the fluorescence-labeled PCR products were directly hybridized on the microarray. Hybridization signals were obtained with an ordinary fluorescent microscope. The sensitivity of the system for virus detection reached 10(2) copies/microL. With the help of inner controls, the system provided reliable results without false negatives and false positives. CONCLUSIONS: The microarray-in-a-tube system is a rapid, labor-saving tool for multiple virus detection with several advantages, such as convenience, prevention of cross-contamination of the PCR products, and potential for multiple-gene detection.

DNA Probes↗

PubMed2006

Detection of fetal DNA in maternal plasma by microarray coupled with emulsions PCR.

BACKGROUND: The presence of fetal DNA in maternal plasma made non-invasive prenatal diagnosis possible. Although fetal DNA has been used in several genetic disease diagnoses, many challenges remained in the detection methods. We attempted to develop a sensitive and reliable microarray coupled with emulsions PCR method to detect the fetal DNA in the plasma of pregnant women. METHOD: Fetal DNAs extracted from the plasma of pregnant women were amplified in emulsions, and fluorescence was labeled at the same time. The labeled target DNAs were hybridized and detected by the capturing DNA probes on a modified slide. Six Y chromosome special sequences in gene of SRY, DYS and DYZ as the marker of fetal DNAs were detected simultaneously in this study, and the beta-globin gene was detected as marker of total DNA from maternal plasma. An unrelated sequence was also detected as negative control in this study. 76 pregnant women in the first trimester of gestation joined in this study. Conventional PCR and real time PCR were also carried out for comparison. RESULTS: We could detect the fetal DNAs reliably with this method in early stage of gestation. The 6Y chromosome sequences were detected in 40 of the 42 male fetus carrier samples, and no Y special sequence was detected in the female fetus carrier samples. The earliest plasma sample which we could detect in this study was collected on the 31st day after pregnancy. CONCLUSIONS: The results suggest that the microarray coupled with emulsions PCR method could be used for fetal DNA detections, and emulsions were useful in DNA amplification as reaction media to overcome the primer incompatibility which frequently encounters in multiplex PCR amplification. Our methods have the potential in high-throughput assays and could be widely used in clinical researches and diagnosis.

DNA↗

PubMed2006

Evaluating the binding affinities of NF-kappaB protein to the single-nucleotide mismatch DNA binding sites by using double-stranded DNA microarray.

Protein-DNA sequence-specific interaction plays an essential role in many biological processes. Here we immobilized a series of double-stranded DNA probes on an agarose coated slide to investigate the binding affinity of NF-kappaB p50 homodimer to the single-nucleotide mismatches (G<-->A or T<-->C) of the 10 base pair (bp) protein binding sites. The results demonstrated that the nucleotides at different positions contribute differently to the p50p50/DNA binding interaction. Within the 10 bp binding sites, the 5tG or 6cA mismatch has less effect on the protein-DNA binding affinity. Even the 5tG mismatch may have the ability to enhance the protein-DNA interaction (5t/w = 1.07). On the other hand, the 7cA or 10tG mismatch blocked the protein-DNA interaction more significantly than other six single-nucleotide mismatches. (7c/W = 0.37, 10t/W = 0.35). It also indicated that the duplex DNA probes immobilized on the agarose-coated surface were apt to be recognized by DNA-binding proteins, and this method would provide a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.

Base Pair Mismatch↗

PubMed2005

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays.

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiments showed that the elongation temperature of 50 degrees C and the Mg(2+) concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.

Carbocyanines↗

PubMed2005

A semi-quantitative microarray method to detect fetal RNAs in maternal plasma.

OBJECTIVES: To set up a semi-quantitative microarray method for the detection of fetal RNAs in maternal plasma. METHODS: We developed a semi-quantitative microarray method for the detection of placental RNA in maternal plasma. Firstly, the selected fetal RNAs were linearly amplified from the maternal plasma and then fluorescently labeled as the target DNAs. Finally, the targets were hybridized and detected by capturing DNA probes on a microarray slide. Two genes of beta subunit of human chorionic gonadotrophin (beta-hCG) and zinc finger gene on the Y chromosome (ZFY) were assayed with the microarray, and beta actin gene was used as an internal standard. Eighty-five pregnant women in the first trimester and the third trimester of gestation joined in this experiment, 14 of them also sampling in 36 h after delivery for the same assay. Real-time quantitative PCR was performed for comparison. RESULTS: It was found that the mRNA level of beta-hCG decreased with the increasing of gestation age, and it was much higher in the carriers of the female fetus than in the carriers of the male fetus in the first trimester of gestation, which was consistent with the real-time quantitative PCR results. The results also reveal that delivery would result in the clearance of fetal mRNA in maternal plasma. CONCLUSIONS: The results suggest that the semi-quantitative microarray method has great potential as a high-throughput assay in prenatal diagnosis and clinical laboratory.

Chorionic Gonadotropin, beta Subunit, Human↗

PubMed2005

A free-labeled method for DNA-binding protein detection using a double-stranded DNA microarray.

We have developed a new type of double-stranded DNA microarray to perform detection of sequence-specific DNA-binding proteins. The DNA-binding site of a DNA-binding protein is divided into two fragments. One fragment was immobilized on an aldehyde-coated glass microscope slide surface via chemical bonds. The other fragment was labeled with a fluorescent molecule. When using this kind of double-stranded DNA microarray, the labeled DNA fragment was pre-incubated with detection sample for 5 to 10 minutes and then hybridized with the microarray. In our experiment, six different concentrations of Nuclear Factor kappa-B P50 homodimer in detection samples were tested. The microarray fluorescence intensity was obtained and the relationship between the intensity and the protein concentration was calculated. The detection results suggested that this free-labeled detection system could have the ability to be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.

Base Sequence↗

PubMed2002

[Genotyping of hepatitis C virus by hepatitis gene diagnosis microarray].

OBJECTIVE: To study the preparation of hepatitis C virus (HCV) diagnosis microarray and its accuracy in diagnosis of gene type of hepatitis C virus. METHODS: Probe and primer and primers were designed in 5'-untranslated region and C region of hepatitis C virus gene. The probes were synthesized by DNA synthesizer. Solutions of probe of the final concentration of 50 micromol/L were made by dissolving the probes into sodium carbonate buffer. Hepatitis C virus genotype array spotting was performed by pin-based spotting robot PixSys5500 with CMP3 pin. The gene chips were prepared by spotting the probes onto the specially treated glass sliders. Sixty HCV RNA positive serum samples were obtained from the in-patients of the Nanjing Second Hospital (experimental group), and 60 HCV RNA negative serum samples were obtained from the healthy people undergoing physical examination (control group). Quantitative examination of serum HCV RNA was made by fluorescent quantitation PCR. The HCV RNA in the serum specimens of the experimental group (with the HCV RNA concentration of more than 500 copies/ml) and of the control group (with the HCV RNA concentration of less than 500 copies/ml) was isolated and purified, underwent reversed transcription and nested PCR to be amplified, and then genotyped by gene microarray and HCV RNA sequencing. During the experiment, double blind method was used. RESULTS: Tested by the gene microarray, the serum specimens in the experimental group were all HCV RNA positive, out of which 46 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, 2 cases were 2b type, 2 cases were 1b + 2a type, and 2 cases were 3a type. Tested by nucleotide sequencing assay, 50 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, and 2 cases were 2b type. The double-blind test results showed a coincidence rate of 93.3% in genotyping HCV by these two methods. CONCLUSION: Hepatitis gene microarray can be used in detection of serum HCV RNA and in diagnostic genotyping with great accuracy.

Genotype↗

PubMed2002

Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film.

We developed a new technique to immobilize a set of molecular beacons on an agarose film-coated slide and found that it has the ability to identify a single nucleotide difference in label-free DNA targets. The annealing properties, specificity and hybridization dynamics of the present technique were compared with those of the conventional technique that directly immobilizes molecular beacons on a planar glass slide. It is demonstrated that the molecular beacon array on an agarose film has high quench efficiency, an excellent discrimination ratio for single nucleotide mismatches and a short detection time. We hypothesize that such a low fluorescence background and high specificity molecular beacon array will find practical applications in label-free, high-throughput mutation analysis and disease diagnosis.

Base Sequence↗