Massively parallel signature sequencing
Approaches that can be used to identify the mRNA transcripts present in a sample as in (SAGE), is quantified continuous analysis of gene expression, parallel signature sequencing large (MPSS), the biochemical The sequenced operations Na was changed significantly sequencing techniques. Treatment of Lynx, a company that was founded in 1992 by Sam Eletr and Sydney Brenner, (MPSS) or next-generation sequencing technology first, massively parallel signature sequence, was developed in 1990. The MPSS, read the sequence of steps of the four nucleotides, bead-based method, was used for a comprehensive approach followed by adapter ligation adapter decode. Loss and displacement sequence-specific of a particular sequence, this method is more susceptible to it. Because technology is very complex, MPSS was was sold in a laboratory that is independent of “insider” the only treatment of Lynx, from inorganic DNA sequencing. Lead treatment of Lynx, the development of the synthetic sequence, the merger (Illumina later acquired) and Solexa in 2004, a simple approach was obtained from Manteia prediction Medicine MPSS is out of date. However, the main characteristics of the MPSS output were typical of Save “next generation” data type that contains hundreds of thousands of short DNA sequences. In the case of MPSS, usually, they are used for DNA sequencing on the measurement of gene expression.
By counting the number of mRNA molecules each derived from any gene, massively parallel signature sequencing (MPSS) has an open platform for analyzing gene expression levels in a sample. The PCR products of DNA derived from this place, so as to obtain a 100,000-PCR product in a unique tag, each mRNA molecule corresponding is amplified. Tag is used to attach the PCR product fines. It is determined by each band after several rounds of sequencing of the ligation base using a signature sequence type II restriction enzyme BbvI, of 16-20 BP ~, periodically, 17 base pairs of the sequence of high quality can be obtained was. This is done in parallel, approximately one million sequences signature is prepared for a single experiment.
The 17-20 (base pairs) can be can be identified by generating a base, MPSS is the 3 ‘end of 3’ at the site of (DpnII or generally Sau3A) restriction enzyme specific transcripts of mRNA array signature near. Signature sequence is cloned to one million particles. Technique ensures that one type of DNA sequence is microbeads. Copied 50 Special Report in a biological sample if it exists, particles 50 different, for 100,000 copies of the signature of specific sequences were amplified These transcripts are captured on each particle Farm You. Then, fine particles, is placed in the quantification and flow cell sequencing. I will decode the sequence signature of parallel identification of the four bases by hybridization encoder that is fluorescently labeled. I have a unique label that was detected after hybridization by the image array of micro-beads of each encoder. The sets of four bases to be removed by dividing, for a new round of hybridization image acquisition and coding, the next step is to clarify the four bases below. Raw output is a list of signature sequences of 17-20 base pairs can be identified by the human genome for gene identification.
Signature sequences MPSS data within (MPSS tag) are analyzed and compared to the signature of all other same signature are counted all. The expression level of each gene was calculated by dividing the number of signature of this gene by the total number of signatures of all mRNA present in the data. This means that it is possible to combine data sets of multiple assays of mRNA samples on the same initial, MPSS data set is summed in nature. To simplify the data analysis routine sensitivity levels, management and cell number per molecule of RNA, the MPSS sets of data in digital form. was cloned into the micro-beads of using the Lynx Megaclone technology cDNA fragment. Each, including starting with one million mRNA molecules from the cell or tissue sample, 100 000 copies of the cloned DNA Megaclone, 100 万 beads, from RNA molecules, each of which is generated. The covalent end, as is available for DpnII sequencing reactions, all molecules are attached to microbeads part thereof of poly (A). Specificity long tag sequence, high 9-10 BP classic label SAGE can be obtained. Level of gene expression unique is represented by the number of transcripts present in the molecule of one million output SAGE similar. An important advantage is the size larger than the library SAGE. Typically, the library has a property 1000000 MPSS tag about 20 times greater than the library SAGE. Not only due SAGE MPSS Related to the lack of ambiguity and restriction enzyme recognition sites in annotation tag, some of the drawbacks associated with the loss of transcript few. Absolute gene expression and high sensitivity prefer certain MPSS. However, this technology is available via a single Lynxgen Pew Ltd.