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Lecture – FALL 2017 Microarray Analysis and Omics Technology
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Introduction Approximately humans have genes Only a fraction of these are actively expressed as mRNAs at any one time. Hybridization between the cDNA reverse transcribed from a biological sample to a pre-designed complementary DNA probe arranged on a slide, or array, is the basis of DNA microarrays.
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A microarray therefore consists of a pre- designed library of synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix. In microarrays fragments are attached to a substrate and then probed with a known gene sequence.
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How arrays are made? Some methods basically use a robot to “print” pre- designed probes that have been attached to fine needles onto a chemical matrix surface using surface engineering (examples include fine- pointed pins, needles and ink-jet printing). Other methods employ photo-activated chemistry and masking to synthesize probes one nucleotide at a time on a solid surface in repeated steps to build up probes of specific sequence in designated locations.
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DNA Microarrays Show Differences in Gene Expression
Microarray chips contain fragments from genes in the group to be analyzed Full genome of bacteria or yeast, or protein families from larger genomes mRNA or cDNA from different samples are differentially tagged Analysis on the same chip shows differences
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Transcriptomics Transcriptomics uses high-throughput techniques based on DNA microarrays For further details about microarrays see Lucchini et al., Microbiology, 147, (2001)
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Transcriptomics
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Transcriptomics Nelson & Cox, “Lehninger, Principles of Biochemistry”, 4th edn, 2004, p. 328 SYLICA 'Omic Technologies – Bowater Feb 2013
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Transcriptomics Experiments performed under different conditions
Determines effect of conditions on expression Produces huge amount of data Lots of repeats required - expensive
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Isolate and purify mRNA from samples of interest.
A basic protocol for a DNA microarray is as follows: Isolate and purify mRNA from samples of interest. Since we are interested in comparing gene expression, one sample usually serves as control, and another sample would be the experiment (healthy vs. disease, etc)
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Reverse transcribe and label the mRNA.
In order to detect the transcripts by hybridization, they need to be labeled, and because starting material maybe limited, an amplification step is also used. Labeling usually involves performing a reverse transcription (RT) reaction to produce a complementary DNA strand (cDNA) and incorporating a florescent dye that has been linked to a DNA nucleotide, producing a fluorescent cDNA strand. Disease and healthy samples can be labeled with different dyes and cohybridized onto the same microarray in the following step.
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Hybridize the labeled target to the microarray.
This step involves placing labeled cDNAs onto a DNA microarray where it will hybridize to their synthetic complementary DNA probes attached on the microarray. A series of washes are used to remove non-bound sequences.
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Scan the microarray and quantitate the signal.
The fluorescent tags on bound cDNA are excited by a laser and the fluorescently labeled target sequences that bind to a probe generate a signal. The total strength of the signal depends upon the amount of target sample binding to the probes present on that spot. Thus, the amount of target sequence bound to each probe correlates to the expression level of various genes expressed in the sample. The signals are detected, quantified, and used to create a digital image of the array.
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Omics stands for genomics, proteomics or metabolomics.
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Aim Characterization and quantification of pools of biological molecules that translate into the structure, function, and dynamics of an organism or organisms. A critical part of System and Functional biology Functional genomics aims at identifying the functions of as many genes as possible of a given organism. It combines different -omics techniques such as transcriptomics and proteomics with saturated mutant collections. Systems biology is the computational and mathematical modeling of complex biological systems. It is a biology-based interdisciplinary field of study that focuses on complex interactions within biological systems.
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Molecular biology: major scientific discipline for past ~50 years
Genomics = “analysis of genomes”: became important science during 1990’s Analyses of various other biological molecules have developed into their own scientific disciplines; e.g. Metabolomics = “analysis of metabolites”, etc. Transcriptomics/Proteomics: developed during past years Bioinformatics: has developed as major branch of science - enables efficient analysis of data from “omics” experiments
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Genomics & Technology Significance of “omics” coincides with dramatic improvements in different technologies: molecular biology: increased range of approaches for purification and manipulation of proteins and nucleic acids computers: required for gathering and analysis of data internet: allows data to be shared, quickly and easily All developments have increased speed and cost-effectiveness - available to much wider audience
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Transcriptomes Genome: all of hereditary information encoded in the DNA (or RNA) Transcriptome: set of all mRNAs ("transcripts”) produced from a genome Term can be applied to: complete set of transcripts for a given organism specific subset of transcripts present in a particular cell type or under specific growth conditions Transcriptome varies because it reflects genes that are actively expressed at any given time SYLICA 'Omic Technologies – Bowater Feb 2013
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DNA Microarrays Show Differences in Gene Expression
SYLICA 'Omic Technologies – Bowater Feb 2013
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Polymerase Chain Reaction (PCR)
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Adaptations to PCR Reverse Transcriptase PCR (RT-PCR)
Used to amplify RNA sequences First step uses reverse transcriptase to convert RNA to DNA Quantitative PCR (Q-PCR) Used to show quantitative differences in gene levels
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Proteomes Proteome: set of all proteins produced under a given set of conditions Term can be applied to: complete set of proteins for a given organism specific subset of proteins present in a particular cell type or under specific growth conditions Proteome varies because it reflects genes that are actively expressed at any given time Proteomics analyses many samples using 2D-electrophoresis and mass spectrometry High-throughput, but less than transcriptomics SYLICA 'Omic Technologies – Bowater Feb 2013
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Gel Electrophoresis Electrophoresis separates molecules by size
Resolution is limited Berg, Tymoczko & Stryer, “Biochemistry”, 6th edn, 2006, p. 71
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Isoelectric Focusing Electrophoresis across a pH gradient
Proteins migrate to their isoelectric pH Berg, Tymoczko & Stryer, “Biochemistry”, 6th edn, 2006, p. 73 The isoelectric point (pI, pH(I), IEP), is the pH at which a particular molecule carries no net electrical charge in the statistical mean. SYLICA 'Omic Technologies – Bowater Feb 2013
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Ampholyte an electrolyte that can either give up or take on a hydrogen ion and can thus behave as either an acid or a base.
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Two-dimensional Gel Electrophoresis
Protein sample initially fractionated in one dimension by isoelectric focusing SDS-PAGE performed perpendicular to original direction Separates proteins according to pI and mass Berg, Tymoczko & Stryer, “Biochemistry”, 6th edn, 2006, p. 74 SYLICA 'Omic Technologies – Bowater Feb 2013
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Two-dimensional Gel Electrophoresis
Proteins from E. coli separated by 2D-electrophoresis >1,000 proteins can be resolved Berg, Tymoczko & Stryer, “Biochemistry”, 6th edn, 2006, p. 74 SYLICA 'Omic Technologies – Bowater Feb 2013
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Transcriptomics v Proteomics
Transcriptomics and proteomics are both very powerful Differences in their practical application: Transcriptomics is robust, relatively cost-effective and user-friendly Proteomics still relatively limited – problems can remain with purification and stability of proteins
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Bioinformatics: Mining the Data
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NCBI: Eukaryotic Genomes
SYLICA 'Omic Technologies – Bowater Feb 2013
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NCBI: Microbial Genomes
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NCBI: Microbial Genomes
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Links to brief description of all resources at NCBI
NCBI – Useful Links Links to brief description of all resources at NCBI
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Databases Summary Many databases are available
- some have lot of general information (NCBI, EBI) - some have specific data (Pfam, SWISS-PROT) - some relate to specific research interests (TAIR) Become well informed with specific databases Wide range of databases, web sites and other resources are available for in silico analysis of biological data Great advantages, but beware of potential pitfalls – understand capabilities and limitations! Use information intelligently: always ask if the conclusions make biological sense may require further analyses or experimentation
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“Omics” Overview Analyses of various biological molecules have developed into their own scientific disciplines; e.g. Metabolomics = “analysis of metabolites”, etc. Transcriptome: set of all mRNAs ("transcripts”) produced from a genome Proteome: set of all proteins produced under a given set of conditions Both can vary because they reflect genes that are actively expressed at any given time Transcriptomics and proteomics are both powerful, but are used differently: transcriptomics is cheaper and more user friendly than proteomics
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