Chapter 1. from genomics to proteomics Ⅱ

Transcription

1 Proteomics Chapter 1. from genomics to proteomics Ⅱ 1

2 Functional genomics Functional genomics: study of relations of genomics to biological functions at systems level However, it cannot explain any more than individual parts of the biological systems Two powerful technological tools -Large-scale mutagenesis -RNA interference (RNAi) 2

3 Large-scale mutagenesis To establish the function of a gene, one can mutate it and observe the effect on phenotype. Two approaches: genome-wide mutagenesis by homologous recombination (gene knockout) -> for small genomes genome-wide random mutagenesis by irradiation -> can produce more subtle point mutations 3

4 RNA interference (RNAi) For functional genomics, RNAi is useful because the introduction of a double-stranded RNA homologous to an endogenous gene results in the rapid destruction of any corresponding mrna => silencing of that gene. 4

5 Transcriptomics Transcriptomics: study of mrna expression profiles based on DNA sequencing (genes are turned on/off depending on the condition and environment) -sequence sampling : (from slide no. 23) -cdna microarray is a typical tool for transcriptomics (1) mechanical spotting of cdna molecules (2) in situ oligonucleotide chip 5

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7 Sequence sampling techniques for Global analysis of gene expression (1) Random sampling of cdna libraries (2) Analysis of EST (expressed sequence tag) databases (3) Differential display PCR (4) Serial analysis of gene expression (SAGE) (5) Massively parallel signature sequencing (MPSS) 7

8 Serial Analysis of Gene Expression (SAGE) -B : Biotin -Pink circle :Streptavidin-coated beads -Linkers A & B : differ in sequence except that have 3 CATG hanging -Downward triangle : recognition site for anchoring enzyme (NlaIII) - Extended arrow : recognition site for type IIs restriction enzyme (tagging enzyme) 8

9 Need for proteomics Transcriptomics, mutagenesis, RNAi dominated functional genomics because of the technology used; high-throughput clone generation and sequencing. Nucleic acids are only information carriers. We can only indirectly infer protein functions from these studies. Proteins are the building block of body and biological functions. Thus, direct study of proteins is needed. 9

10 Importance of proteomics (1) (1) The function of a protein depends on its structure and interactions. (2) Mutations and RNA interference are coarse tools for large-scale functional analysis. (3) The abundance of a given transcript may not reflect the abundance of the corresponding protein (4) Protein diversity is generated post-transcriptionally (alternative splicing). 10

11 Importance of proteomics (2) (5) Protein activity often depends on post-translational modifications. (6) The function of a protein often depends on its localization. (7) Some biological samples do not contain NA s. (8) Proteins are the most therapeutically relevant molecules in the body. 11

12 Scope of proteomics (1) (1) Sequence and structural proteomics: databases -protein sequencing (chap3), bioinformatics (chap5), storage, presentation, comparison and prediction of structure (chap6) (2) Expression proteomics: microarrays -Fig 1.9; 2D-GE (chap2), Protein quantitation (chap4), analysis of post-translational modification (chap8), use of protein chip for analysis and quantitation (chap9) 12

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14 Scope of proteomics (2) (3) Interaction proteomics -Fig 1.10; yeast two hybrid system (chap7) (4) Functional proteomics: direct test of proteins High-throughput functional analysis (chap9) 14

15 Challenges of proteomics No single technique is suitable for every application Lack of an amplification method such as PCR for NA s 15