Study Of The Detection Assay For Biomarkers

Biomarkers are those which can reflect the interaction of biosystem with chemical, physical substances or biological factor in enviroment. They can be divided into three categories: (1) Biomarker of exposure, which indicates those inflecting the concentration of exogenenous materials or their metabolites, or the concentration of the products from the interaction of exogenenous material with some target cells. Internal dose ones and external dose ones are concluded. (2) Biomarker of effect. It indicates those indicaters inflecting the biochemical, biophysical, behavioral, or else changes. Three types of biomarkers, early biological effect, altered structure / functon and disease, are comprised. (3) Biomarker of susceptibility, namely the markers of inherent or acquired ability responsing to exogenous materialsAmong the present biomarkers, single nucleotide polymorphisms ( SNPs ) and proteins are the most commen. This study is aiming to develope new detection assays for SNPs and protein, which were mainly the discovery of the human genomic research.SNPs are the most abundant form of genetic variation in the human genome. SNPs are DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is altered. Each person has his own set of SNPs which determine the personality of people, and result the genetic diversity of huaman. SNPs have a major impact on how humans respond to disease; environmental insults such as bacteria, viruses, toxins, and chemicals; and drugs and other therapies. This makes SNPs of great value for biomedical research and for developing pharmaceutical products or medical diagnostics.Thus, how to discove, detect, research and utilize SNPs with high-throughput assays attracted the attentions of scientists and company. The discovery of more and more protein markers is attributed to protomics. After the completion of Human Genome Project(HGP), one of the important missions of life science is to explain and vilidate the function and regulation of genes. This period was called Post-genome era. Since the function of genes is expressed by proteins encoded by genes, proteins are the real performer of life activities, and proteomics is consider the most important part of the post-genomic investigation. One of the important tasks is to study disease-related proteins. Through the proteomics research, a large amounts of disease-related proteins markers were found and used in medical practices. With the developing of proteomics research, thousands of proteins with potential diagnostic and/or therapeutic applications are expected to emerge from the various genome projects. Proteins in different tissues or individual cells can be up- or downregulated in response to internal or external stimuli, signal transduction, transcriptional control, medication, disease, or pathogens. Because the protein markers related to early stage of disease are within the very low concentration, to develope Ultrasensitive detection assay is of utmost importance not only for proteomics research but also for diagnostic and technological purposes.Part One Genotyping Single-nucleotide polymorphism using ligation mediated SAGE technologyThere are many detection methods for SNPs, the focus about the methodology of the detection methods for SNPs is on developing high-throughput assay on large-scale SNPs to discover, detect, research and apply SNPs. The present high-throughput ananytical assays for SNP include gene chip, Fluorescence resonance, Fluorescence polarization, Pyrosequencing, denaturing-high performance liquid chromatography ( DHPLC), Bead array and mass spectrometry. Gene chip and bead array are the common methods for the most largest scale SNPs detection, the detection of mid-scale SNPs is mainly depended on (matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF) and Fluorescence polarization genotyping assay based on primer extension assay. But each of them has its own shortages. For example, GeneChip and BeadArray suffer from the limitation in fabrication and design, mass spectrometry assay require costly instrument and skilled operater. The results of Fluorescence Polarization Assay are easily affected by non-specific products. So, we pursue to establish a new platform for detection of SNPs.A new platform was set up by taking use of SAGE (serial analysis of gene expression) technology to set up with high-throughput for SNPs detection. This method was called LM-SAGE.The outlined test procedure is as follow. First, the mismatch DNA heteroduplexes formed by wild-type and mutation DNA was cleaved by CEL I nuclease in mismatched sites to generate a sticky double-strand. After the sticky double-strand was linked to a linker, the 17bp tags contained mismatched sequence were obtained by digestion of restriction endonuclease Mme I, and then the tags were concatenated and cloned for sequencing. We can identify the locations of SNP sites according to the sequences of tags.What the results are : (Ⅰ) All 8 mismatches were detected by LM-SAGE simultaneously; no prior knowledge about the SNPs contex was needed. (Ⅱ) the minimal amount of start sample available to located all mismatches formed by SNPs in targeted samples was 2.4×106 molecules; (Ⅲ) Dilution experiments were performed to test for the ability to detect mutations in the presence of an excess of wild-type DNA. In 1-10 fold excess of wild-type DNA sample, all eight mismatches were detected. In 20-fold one, only A/A and A/G mismatch were detected.The conclusion indicated that LM-SAGE is easy-to-use. It dosen't require any other expensive instrument if couple to using commercial sequencing. It can detect known SNPs, discove unknown SNPs, and locate the SNPs at the same time. LM-SAGE is a novel high-throughput method for SNPs detection.This study is the first to utilize SAGE (serial analysis of gene expression) technology to detect SNPs. Comparing to other methods, It is easy to use and dose not require any expensive instrubent if coupled to commercial sequencing service. And it require no sequence information of taget sample to detect and locate SNPs. It would be value to studying and utilization of SNPs if this method reported here is investigated further and generalized. Part Two Ultrasensitive colorimetric detection of protein by nanoparticle-modified aptamerThe traditional methods for protein detection, such as ELISA or Western Blot (WB) can't meet the demand of protein marker recognition. The current ultrasensitive protein detection is mainly based on the use of antibody, and aptamer research is still in its infancy. Aptamer are RNA or DNA oligonucleotides with the properties binding to given ligands such as proteins with specifity and high-affinity, which is similar to, sometimes stronger than, antibody. So, aptamer can be the valuable tools for analyse proteins. Among the present methods for protein detection using aptamers, some of them are with high sensitivity, some with high sensitivity but require expensive instrument. This study takes use of aptamer modified by Au naloparticle to construct a new method for protein detection. This methos can fastly detect proteins with high sensitivity compairing to other methods, and requires no expensive instruments. It has wide application prospect and large market value.THE objection of this study is to establish a new method for protein marker detection. The outlined methodology is as follow: first, target protein was bound to nanoparticle-modified aptamer and another aptamer probe modified with biotion to form a sandwich complex. And then, the sandwich complex was captured in microwell by avidin. After amplification with silver-enhancement, the absorbance resulted from aptamer-protein complex was detected by colorimetry. The platelet-derived growth factor B-chain (PDGF-BB) was used to assess the sensitivity and limit of detection.What the results are: (Ⅰ)In the range of 1fM-1μM, the absorbance induced by the concentration of PDGF has dose dependence. R2 = 0.9801. (Ⅱ)The minimal detection limit was 1 fM targeted protein. (Ⅲ)It could detect the proteins biological samples.Conclusion: this new NP-modified aptamer assay could offer an ultrasensitive detection of protein. It requires no complicated or costly instruments and is easy-to-use. It has a good future for detecting ultratrace levels of disease-related protein markers that cann't be measured by conventional methods.