Huafang Gao

Comparison of Different Methods for Preparing Single Stranded DNA for Oligonucleotide Microarray

Abstract Single stranded DNAs (ssDNA) were crucial reagents for nucleic acid analysis on oligonucleotide microarrays. Methods for preparing ssDNAs have been reported, but the systematic study about them was still lacking now, especially for oligonucleotide microarrays. Using oligonucleotide microarrays, some of the most popular methods were compared in this report, including asymmetric polymerase chain reaction (PCR), magnetic beads based method, denaturation of double stranded DNA (dsDNA) by heating or by alkaline treatment. All the ssDNA targets prepared by the four methods were applied to oligonucleotide microarrays. The results indicate that the ssDNA targets generated by asymmetric PCR and magnetic beads based method have shown high sensitivity and specificity in hybridization, but those generated by denaturation of dsDNA via heating or alkaline treatment produced serious false negative signals under the same hybridization conditions. Considering the simplicity and the cost of the method, asymmetric PCR is a more favorable approach for oligonucleotide microarrays. In view of the fact that most of the present asymmetric PCR procedures were in two steps, and a purification process was necessary before the second step amplification was carried out, a one-step asymmetric PCR was attempted in this work. The results demonstrate that it was a preferred approach for ssDNA preparation for oligonucleotide microarrays use.

Lectin RCA-I specifically binds to metastasis-associated cell surface glycans in triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential.MethodsWe used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I.ResultsUsing the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.ConclusionsWe discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827

The design and application of DNA chips for early detection of SARS-CoV from clinical samples

Abstract Background: SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test. Objectives: Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method. Study design: We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection. Results: A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Prenatal diagnosis of β-thalassemia by chip-based capillary electrophoresis

To explore the potential of chip‐based capillary electrophoresis for rapid prenatal diagnosis of genetic diseases, we developed a prenatal diagnosis method for β‐thalassemia, one of the most common recessive inherited disorders.

Probe selection algorithm for oligonucleotide array-based medium-resolution genotyping.

Medium-resolution genotyping has the goal of distinguishing different subgroups instead of each element in a group. An oligonucleotide array provides an inexpensive, high-throughput method to identify differences in DNA sequence among individuals, which is fundamental for genotyping. As the cost and difficulty of designing and fabricating the oligonucleotide array dramatically increase with the number of probes used, it is therefore important to have a design with a minimum number of probes meeting the requirement of medium-resolution genotyping. The first algorithm for designing and selecting probes for oligonucleotide array-based medium-resolution typing is reported. The goal in deriving the algorithm was to select a minimum number of probes from a large probe set on the premise of minimum loss of resolution. The algorithm, which was based on entropy, conditional entropy and mutual information theory, was used to select the minimum number of probes from a large probe set. The algorithm was tested on a human leukocyte antigen (HLA) sequence data set. Thirty probes were selected from 390 probes for HLA-A, and 60 probes were selected from 767 probes for HLA-B. Although the number of probes was reduced by almost ten times, the distinguishability was reduced only a little, by 0.45% (from 99.90% to 99.45%) for HLA-A and 0.27% (from 99.84% to 99.57%) for HLA-B, respectively. This is a satisfactory and practical result.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb–host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF‐κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb‐host interactions.

Mutation spectra and founder effect of TMC1 in patients with non-syndromic deafness in Xiamen area, China

To analyze the spectrum and founder effect of TMC1 mutations in patients with non‐syndromic deafness in the Xiamen area. Sporadic pedigrees were detected by targeted next‐generation sequencing, and 110 unrelated patients from Xiamen Special Education School were analyzed through Sanger sequencing for the TMC1 gene. In total, 53 SNPs were designed to analyze the haplotypes of the TMC1 c.2050G>C mutation. The probands of three families were found to be homozygous for TMC1 c.2050G>C, and their parents were all heterozygous for the TMC1 c.2050G>C mutation. In 110 unrelated patients from Xiamen Special Education School, four were found to carry compound heterozygotes of TMC1 c.2050G>C, which were compound heterozygotes of c.804G>A, c.1127T>C, c.1165C>T, and c.1396_1398delAAC, respectively. Three types of TMC1 polymorphisms (c.45C>T, c.1713C>T, c.2208+49C>T) and two heterozygotes of novel variants (c.1764‐4C>A, c.2073G>A[p.K691K]) were found in the remaining 100 patients. In total, four novel variants were detected in this study. These mutations and variants were not detected in 100 normal samples. The haplotypes of the probands of families with TMC1 c.2050G>C were identical. There were unique hotspots and spectra of TMC1 mutations in the Xiamen deaf population. Haplotype analysis is useful to understand the founder effect of the hot spot mutation.

The polymorphisms of intron1 sequences of HLA-A and -B

Human leucocyte antigen (HLA) class I antigens are highly polymorphic membrane glycoproteins present on most nucleated cells. The polymorphism region is mainly located in exons 2 and 3 of HLA class I genes, which is flanked by introns 1 and 3. Thus, the sequence information of introns 1 and 3 is important for the genotyping of HLA. However, the information about them has not been extensively reported by now. In this work, the intron 1 sequences of HLA-A and -B of 51 standard genomic DNAs provided by the 13th International Histocompatibility Work Group (IHWG) were cloned and sequenced. The polymorphism of the intron sequences were also analyzed by the software Clustal W 1.82. Some sequences were chosen to compare with the standard sequences published in National Center for Biotechnology Information (NCBI). The comparison showed that all the sequences matched exactly with the standard sequences. The knowledge of intron 1 sequences could be very important not only for developing DNA-based typing strategies for the HLA-A and -B alleles but also for establishing an understanding of the evolutionary mechanisms involved in the polymorphism generation of HLA class I alleles.

A novel approach for quality control of oligonucleotide probes using DHPLC

Abstract Oligonucleotide microarray has become one of the most powerful tools in the areas such as genotyping, gene expression, and SNPs detection. However quality control is critical for rapid development of this technology. Several approaches have been attempted to monitor the fabrication steps from oligonucleotide probe printing to data analysis. Yet, quality control methods for presynthesized oligonucleotide probes have not been available. Here we propose a novel approach for the control of the quality of oligonucleotide probes using the technology of denaturing high-performance liquid chromatography (DHPLC). In this work, all possibly existing types of poorly synthesized oligonucleotide probes prepared by artificial method were used to validate the discriminating ability of DHPLC. It was found that DHPLC can not only provide the purity ratio of the oligonucleotide probes accurately, but also detect misincorporation of chemicals in the oligonucleotides such as one base substitution, one base deletion, or failed amino modification which is difficult to be distinguished through routine methods such as PAGE. For 277 oligonucleotide probes, 31% of the probes had a purity ratio of 70% or less. The results indicate that DHPLC can be a practical approach to control the quality of oligonucleotide probes before they are printed to the microarray slides.