No abstract available.
DNA* ; Nucleic Acid Hybridization*

Gene Library ; Humans ; Nucleic Acid Hybridization ; methods ; Pulmonary Fibrosis ; diagnosis

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.
Nucleic Acid Hybridization ; Nucleic Acid Probes ; Oligonucleotide Array Sequence Analysis ; methods ; Peptide Nucleic Acids ; genetics ; Sensitivity and Specificity ; Surface Plasmon Resonance

BACKGROUND: The most consistent chromosomal abnormality in ependymomas, is loss of 22q (17-75%) and gain of 1q (0-50%). However, significance of this abnormality is uncertain. METHODS: Genomic imbalances in 27 Korean ependymomas, including 21 low grade ependymomas, 4 anaplastic and 2 myxopapillary ependymomas, were analyzed by degenerate oligonucleotide primed-PCR-comparative genomic hybridization. RESULTS: Common gains were found in 17 (63%), 20q (59%), 9q34 (41%), 15q24-qter (33%), 11q13 (30%), 12q23 (26%), 7q23-qter (26%), 16q23-qter (30%), 19 (26%), and 1q32-qter (22%). DNA amplification was identified in 12 tumors (44%). Chromosomal loss was a less common occurrence in our study, but was found in 13q (26%), 6q (19%), and 3 (11%). CONCLUSION: The recurrent gains or losses of the chromosomal regions which were identified in this study provide candidate regions that may be involved in the development and progression of ependymomas.
Chromosome Aberrations ; Comparative Genomic Hybridization ; DNA ; Ependymoma* ; Nucleic Acid Hybridization* ; Polymerase Chain Reaction

Carrier State ; Gene Expression Profiling ; Hepatitis B ; genetics ; Humans ; Hybridization, Genetic ; Nucleic Acid Hybridization ; methods

OBJECTIVETo construct a suppression subtractive library of virulence-related genes from c serotype Streptococcus mutans (S. mutans), and lay foundations for screening the virulent genes.

METHODSAfter being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNA was digested with three appropriate four-base-cutting restriction endonueleases to produce fragments of optimal length. The digested DNA of the virulent strain ligated with adaptor was used as tester DNA, and that of the avirulent strain as driver DNA. Then the suppression subtractive hybridization was carried out, and the efficiency of ligation and subtraction detected respectively. The subtracted fragments were inserted into vector pCR2. 1 using T/A cloning kit, and transformed into E. coli TOP10F' competent cells. Those white colonies were selected to construct the suppression subtractive library.

RESULTSAlu I chosen from three restriction endonucleases was verified to be suitable for preparing restriction fragments from S. mutans genomic DNA. Through electrophoresis of Alu I -digested DNA, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency of suppression subtractive hybridization confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene both in tester and driver DNA was later than that in the unsubtracted group by six cycles. It suggested that suppression subtractive hybridization happened indeed. After the subtracted fragments were cloned, 96 colonies were picked up for constructing the suppression subtractive library of virulence-related genes of S. mutans.

CONCLUSIONSuppression subtractive hybridization allows rapid and easy construction of virulence-related gene library of S. mutans.

Escherichia coli ; Gene Library ; Nucleic Acid Hybridization ; Streptococcus mutans ; Subtractive Hybridization Techniques ; Virulence

Genetic Techniques ; Genomics ; Humans ; Nucleic Acid Amplification Techniques ; Nucleic Acid Hybridization ; Sequence Analysis, DNA ; Viruses ; genetics ; isolation & purification

OBJECTIVETo establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD).

METHODSTwelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed.

RESULTSThe amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods.

CONCLUSIONPEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.

Comparative Genomic Hybridization ; methods ; DNA Primers ; Genetic Testing ; methods ; Humans ; Karyotyping ; methods ; Nucleic Acid Amplification Techniques ; methods ; Nucleic Acid Hybridization ; methods ; Oligonucleotides ; chemistry ; Preimplantation Diagnosis ; methods

Peptide nucleic acid (PNA) is a DNA surrogate in which the phosphate deoxyribose backbone of DNA is replaced by repeating N-(2-aminoethyl)glycine units. PNA can hybridize to the complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs. The non-ionic backbone of PNA leads to stable hybrids with the nucleic acids, but at the same time, the neutral backbone results in poor cellular uptake. To address this problem, studies on modified PNA progress rapidly in recent years. We reviewed literature reports combined with our study about the delivery methods, including backbone modified PNA and PNA-ligand conjugates, and the cellular uptake of modified PNA. In addition, we summarized the problems and future prospect of the cellular delivery of modified PNA.
DNA, Complementary ; Drug Delivery Systems ; Glycine ; analogs & derivatives ; Humans ; Nucleic Acid Hybridization ; Oligonucleotides ; Peptide Nucleic Acids ; chemistry ; RNA

BACKGROUND AND OBJECTIVES: The knowledge about chromosomal aberrations manifestated in cancer has been spotlighted recently. The genetic analyses based on the knowledge about chromosomal aberrations are important for the development of diagnotic methods and evaluation of prognostic factors in cancers. Comparative genomic hybridization (CGH) is a powerful tool for evaluating chromosomal aberrations and array CGH significantly enhances these diagnostic effectiveness. The incidence of head and neck squamous cell carcinoma (HNSCC) has been increasing worldwide but the treatment outcomes still have been limited. The aim of this study is to evaluate the location of chromosomal aberrations in HNSCC cell lines with the combination of CGH and array CGH. Materials and MethodZZThe locations of chromosomal aberrations were evaluated in 3 HNSCC cell lines (PCI-1, PCI-13, PCI-50) using the combination of CGH and array CGH. RESULTS: The sites of chromosomal gain shown by CGH in all 3 cell lines were 8q22-qter, 9q32- qter, 10q22, 10q26-qter, 16q12.1-qter, 17p10-p13, 17q21-qter, 19p13.2-pter and 20q. The chromsomal loss found in 2 cell lines were 3p, 4q21-qter, and 18q21-qter. In array-CGH, gained loci were AHRR, MYT1 and PTGIS, etc. Loci of genetic losses were ELAVL4 and GRM7. CONCLUSION: In this study, we identified various genetic gains and losses using CGH and high resolution array-CGH. These data about the patterns of chromosomal aberrations in HNSCC cell lines would be a basic step for understanding more detailed genetic events in the carcinogenesis. CGH combined with array CGH can be a powerful option for transitional oncologic research.
Carcinoma, Squamous Cell ; Cell Line ; Chromosome Aberrations ; Comparative Genomic Hybridization ; Head ; Head and Neck Neoplasms ; Incidence ; Neck ; Nucleic Acid Hybridization