19-09-2012, 04:31 PM
TRANSCRIPTOMICS
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TRANSCRIPTOME
The study of characteristics and regulation of the functional RNA transcript population of a cell/s or organism at a specific time.
Transcriptomics is the branch of molecular biology that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type
Scope
the population of functional RNA transcripts.
the mechanisms that regulate the production of RNA transcripts
dynamics of the trancriptome (time, cell type, genotype, external stimuli)
Central Dogma
mRNA – single stranded RNA molecule
Complementary to DNA
Processed (spliced and polyadenylated) RNA transcript
Carries the sequence of a gene out of the nucleus into the cytoplasm where it can be translated into a protein structure
Massively Parallel Signature Sequencing
Massively Parallel Signature Sequencing (MPSS) is a an open-ended platform that analyses the level of gene expression in a sample by counting the number of individual mRNA molecules produced by each gene.
Hybrid of microarray technology
PRINCIPLE:
cDNA clones can be sequenced by cleavage with typeII restriction enzyme, followed by ADAPTOR annealing
METHODOLOGY:
Tags- 32-mer oligonucleotides sequenced
Complementary “anti-tags” synthesized
Covalently attached to plastic microbeads
Tags mixed with cDNA ligated to form conjugates
Amplified tagged cDNAs were end labeled with a fluorescent probe
Attached to microbeads bearing complementary anti-tags
cDNA cleaved by restriction enzyme DpnII to remove the flourescent label and generate a cohesive site
Initiating primer recognizes DpnII overhang ,and carries recognition site BbvI,cleaves a specific number of bases downstream of recognition site.
Cleaved cDNA is annealed to a set of 16 encoded adaptors and carries a BbvI site, allowing repetition of the process