Transcript Methods
Measurement Methods in Systems Biology Widespread Methods: o GFP Expression o Fluorescence Imaging o Two-Hybrid Screen o Expression DNA Chips o Quantitative PCR o Highly Parallel Sequencing GFP: Green Fluorescent Protein The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. This process takes place when the protein aequorin, also produced by A. victoria, interacts with Ca2+ ions thus emitting a blue glow. The wild-type GFP (wtGFP) from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. One of the most powerful uses of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living organism). Due to this widespread usage different mutants of GFP have been engineered over the last few years: some mutants have been produced with increased fluorescence and the protein major excitation peak has been shifted to 490 nm with the peak emission kept at 509 nm (EGFP). Color mutants have been obtained from the GFP gene as well: in particular the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP) are two colour variants employed for fluorescence resonance energy transfer (FRET) experiments. While most small fluorescent molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. (from: en.wikipedia.org) Neurons expressing GFP The Fluorescence Process Stage 1 : Excitation A photon of energy hEX creates an excited electronic singlet state (S1'). In chemiluminescence, S1’ is populated by a chemical reaction. Stage 2 : Excited-State Lifetime For finite time (typ. 1–10 ns), the fluorophore undergoes conformational changes and is subject to interactions with its molecular environment. (a) the energy of S1' is partially dissipated, yielding a relaxed singlet excited state (S1). (b) not all excited molecules return to the ground state (S0) by fluorescence emission: possibilties are collisional quenching, Fluorescence Resonance Energy Transfer (FRET) and intersystem crossing. Stage 3 : Fluorescence Emission A photon of energy hEM is emitted, returning the fluorophore to ground state S0. Due to energy dissipation (S1’ to S1) the energy of the emission photon is lower (longer wavelength). Difference is called the Stokes shift, making it possible to detect emission photons against the huge background of excitation photons. Fluorescein Fluorescence Microscope http://www.olympusmicro.com/ Two Hybrid Screen Fields & Song, Nature 340,245 (1989) Yeast Two Hybrid Screen: Searching for Protein-Protein interactions Bait Prey Two-hybrid screening is a molecular biology technique used to discover proteinprotein interactions by testing for physical interactions (such as binding) between two proteins. One protein is termed the bait and the other is a prey or library. For the purposes of two-hybrid screening, the transcription factor is split into two separate fragments, called Binding Domain (BD) and Activating Domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for activation of transcription. The key to the twohybrid screen is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in close proximity to each other without direct binding. The most common screening approach is the yeast twohybrid assay. This system utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lacks these nutrients, the yeast fail to survive. In yeast two-hybrid screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain. With a certain bait protein, two hybrid screening can be "directed" to test for protein-protein interaction with a known protein inserted into prey plasmid. Alternatively, "library screening" involves pairing bait protein with millions of different prey plasmids that have been engineered to produce protein from a unique, randomly inserted DNA fragment. (from: en.wikipedia.org) DNA Chips Highly Parallel DNA Detection Methods DNA Detection to… o Define a new ’Microscope’: RNA Profiles of Cells o Test for Virus (AIDS...) o Select medication o Test for GMO o Criminal investigations More Details: http://en.wikipedia.org/wiki/DNA_chip Highly parallel DNA detection I A) Spotted Arrays Rockefeller Highly parallel DNA detection II B) On-Chip Synthesis Affymetrix Highly parallel DNA detection II B) On-Chip Synthesis Affymetrix Mismatch is in center of 25-base probe Highly parallel DNA detection III D) Quantitative PCR (qPCR) Online Detection of Product Standard Curve Quantitative polymerase chain reaction (Q-PCR) is a modification of polymerase chain reaction used to rapidly measure the quantity of a product of polymerase chain reaction. It is preferably done in real-time, thus is an indirect method for quantitatively measuring starting amounts of DNA, complementary DNA or ribonucleic acid (RNA). This is commonly used for the purpose of determining whether a genetic sequence is present or not, and if it is present the number of copies in the sample. (en.wikipedia.org) (c) Roche Highly parallel DNA detection IV C) Massive Parallel Signature Sequencing (MPSS) Individual "cloning" on beads is performed picking a small sample from a large pool of combinations. This ensures that each bead has a unique DNA sequence attached. Brenner et.al. Nature Biotechnology 18:630 (2000) Highly parallel DNA detection IV C) Massive Parallel Signature Sequencing (MPSS) Lynx Brenner et.al. Nature Biotechnology 18:630 (2000) + Searching for unknown sequences Highly parallel DNA detection IV Margulies et.al. Nature 437,376 (2005) Highly parallel DNA detection IV Margulies et.al. Nature 437,376 (2005) Highly parallel DNA detection IV Margulies et.al. Nature 437,376 (2005) Highly parallel DNA detection IV Margulies et.al. Nature 437,376 (2005)