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

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

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

Tandem-repeated sequence of nucleic acid was constructed by splicing 4 fragments which contain the same sequence in the central part, using overlap extension polymerase chain reaction and then repeatedly cloning it in the same vector at different site of restriction endonuclease. Its signal amplification action was evaluated using electrophoresis of hybridized product and dot hybridization assay. 24-repeat sequence was successfully constructed and confirmed by restriction endonuclease digestion analysis. The construct was 25-repeat actually since the vector itself had the same basic sequence. Hybridized product electrophoresis revealed that the 25-repeat sequence could combine with several secondary probes. Dot hybridization assay demonstrated that tandem-repeated sequence was 16-fold more sensitive than that of non-repeated sequence. Tandem-repeated sequence had good effect on signal amplification. It could be easily cheaply prepared in large amount after cloning. Thus, it might be useful in clinical examinations and biological researches.
Genetic Vectors ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Tandem Repeat Sequences ; genetics

OBJECTIVETo construct a subtractive cDNA library of genes transactivated by PS1TP1 protein with suppression subtractive hybridization (SSH) technique.

METHODSSuppression subtractive hybridization technique and bioinformatics technique were used, the mRNA from HepG2 cells transfected with pcDNA3.1 (-) -PS1TP1 and pcDNA3. 1 (-) empty vector was isolated, respectively; cDNA underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library.

RESULTSThe subtractive library of genes transactivated by PS1TP1 was constructed successfully. Sequence analysis was performed in 43 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 12 coding sequences were gotten, which consisted of 10 known and 2 unknown ones.

CONCLUSIONThe obtained sequences may be target genes transactivated by PS1TP1 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some clues for studying the biological functions of PS1T1.

Hepatitis B ; genetics ; Humans ; Nucleic Acid Hybridization ; methods ; Transcriptional Activation ; genetics ; Viral Proteins ; genetics

OBJECTIVETo establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese.

METHODSLabel biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects.

RESULTSThe PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese.

CONCLUSIONThis method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.

Humans ; Nucleic Acid Hybridization ; methods ; Point Mutation ; Reproducibility of Results ; alpha-Globins ; genetics ; alpha-Thalassemia ; diagnosis ; genetics

AIMTo explore a method to replace the isotope probe in the PCR-Select differential screening Kit with DIG labeled probe.

METHODSDifferential expressed sequences isolated from Suppression Subtractive Hybridization (SSH) was translocated onto nitric fibrin film. Probes were prepared with DIG-11-dUTP mixed in by PCR. Hybridization and coloration followed routine operation procedures of DIG labeled probe. Positive hybridization results were verified with reverse Northern blot.

RESULTSThe positive results from the PCR-Select differential screening Kit improved with DIG labeled probe achieved 90% correspondence verified by reverse Northern blot.

CONCLUSIONThe PCR-Select differential screening Kit improved with DIG labeled probe maintained high specificity while avoiding isotope pollution. It can replace isotope probe completely.

DNA Probes ; Digoxin ; Nucleic Acid Hybridization ; Polymerase Chain Reaction ; instrumentation ; methods ; Reagent Kits, Diagnostic

The resource authentication is required for quality assurance and control of Chinese medicine. This review provides an informative introduction to molecular methods used for authentication of Chinese medicinal materials. The technical features of the methods based on sequencing, polymerase chain reaction (PCR) and hybridization are described, merits and demerits and development of the molecular methods in identification of Chinese medicinal materials are discussed.
Biometric Identification ; methods ; Medicine, Chinese Traditional ; standards ; Nucleic Acid Hybridization ; Polymerase Chain Reaction ; Sequence Analysis, DNA

The HLA system was discovered by virtue of the fact that it was polymorphic. The impetus for its discovery was the search for polymorphic antigens to match for transplantation, by analogy with the human red cell blood groups. The most usually DNA method of HLA typing is sequence specific oligonucleotides (SSO) and PCR sequence specific primers (SSP). SSO technique is perfectly suited for analyzing large number of samples, it is not suitable for individual or small numbers. The SSP method is ideal for typing individual samples, but it is costly and requires high capacity thermal cycles for larger numbers of samples. To set up a simple, quick, cheap and high resolution DNA method, were collected sixty-three cord blood samples from Guangzhou Cord Blood Bank, got DNA from blood by the traditional guanidine hydrochloride distillation method. Each sample was simultaneously typed by SSOP, PCR-SSP and reverse dot-blot hybridization (RDB) methods. All of typed is success. The results of three DNA methods are consistent each other. 60 HLA-DRB1 alleles could be accurately distinguished with the RDB method. Our results show that RDB method is a simple, quick, cheap and high resolution method for HLA-DRB types. It can be used in any HLA typing.
Fetal Blood ; immunology ; metabolism ; Genotype ; HLA-DR Antigens ; genetics ; Histocompatibility Testing ; methods ; Humans ; Nucleic Acid Hybridization ; methods