|Massively Parallel Signature Sequencing (MPSS): Cloning of cDNA Fragments on Microbeads|
The MPSS technique occurs in two major steps. In the first step, in vitro cloning of cDNA fragments on microbeads occurs. cDNA fragments are cloned onto microbeads using the Lynx Megaclone technology. Starting with one million mRNA molecules from a particular cell or tissue sample, Megaclone will produce one million beads, each containing 100,000 cloned copies of cDNA from each mRNA molecule. All molecules are covalently attached to the microbeads at their poly(A) ends, so the DpnII end is available for the sequencing reactions. The image on the right illustrates the in vitro cloning of cDNA fragments on microbeads. The procedure involves both microbead tagged vector library preparation and sample preparation.Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine, http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml.
|Massively Parallel Signature Sequencing (MPSS): Sequencing Process and Analysis of Sequence Patterns|
In step two, the microbeads are loaded and the sequencing process is initiated. Typically, five rounds produce about 16-20 signature sequence products. Several rounds of sequence determinations are performed and a sequence pattern or signature is identified from each microbead. The process is performed in parallel with about 1 million sequence signatures produced per overall assay. Each signature sequence is then analyzed, and compared with all other signatures and all identical signatures are counted. The level of expression of any single gene is then calculated.The image on the right illustrates the sequencing process and analysis of sequence patterns. Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine, http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml.The MPSS technique is quite complex. If you would like a more in-depth explanation, please click on the "More Info" button below and access the resources that are provided.
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