Genomics(functional genomics)
- 2. Gene: It is a unit of heridity which is transferred from a parent to offspring and held to determine some characteristic to offspring Genome: The entire set of genetic information in an organism It is encoded in DNA or RNA in case of many viruses coined by HANS WINKLERin 1920 Genomics field of study where entire genome is studied coined by THOMAS RODERICK
- 3. Goals of genomics • Compile the genomic sequences of organisms • Search out the location of the genes for analyzing spatial relationships and annotate the gene set in a genome • Learn the function of genes and their influence • Establish how gene expression profiles of a cell vary under different conditions. • Compare gene and protein profiles among different organisms to learn about evolutionary relationships.
- 4. • The field of genomics comprises of two main areas: 1.Structural genomics 2. Functional genomics Structural genomics • Structural genomics helps to describe the 3- dimensional structure of every protein encoded by a particular genome. • The principal difference between structural genomics and traditional structural prediction is that structural genomics attempts to determine the structure of every protein encoded by the genome, rather than focusing on one particular protein. • This genome-based approach allows for a high- throughput method of structure determination by a combination of experimental and modeling approaches. • experimental methods using genomic sequences • modeling-based approaches .... based on sequence or structural homology of a protein of known structure • or based on chemical and physical principles for a protein with no homology to any known structure.
- 5. Functional genomics • Functional genomics is a field of molecular biology that attempts to describe gene (and protein) functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects (such as genome sequencing projects and RNA sequencing). • Functional genomics is the study of how the genome, transcripts (genes), proteins and metabolites work together to to produce a particular phenotype. • Together, transcriptomics, proteomics and metabolomics describe the transcripts, proteins and metabolites of a biological system, and the integration of these data is expected to provide a complete model of the biological system
- 6. • Functional genomics focuses on the dynamic expression of gene products in a specific context, for example, at a specific developmental stage or during a disease. Why we need to study? • It is estimated that approximately 30% of the open reading frames in a fully sequenced organism have unknown function at the biochemical level and are unrelated to any known gene. • This is why recently the interest of researchers has shifted from genome mapping and sequencing to determination of genome function by using the functional genomics approach. • Example A single gene can give rise to multiple gene products. RNA can be alternatively spliced or edited to form mature mRNA. Besides, proteins are regulated by additional mechanisms such as posttranslational modifications, compartmentalization and proteolysis. Finally, biological function is determined by the complexity of these processes. • The goal of functional genomics is to understand the relationship between an organism’s genome and its phenotype.
- 7. There are several specific functional genomics approaches depending on what we are focused on: • DNA level (genomics and epigenomics); • RNA level (transcriptomics); • protein level (proteomics); • metabolite level (metabolomics). TECHNIQUES • Functional genomics uses mostly multiplex techniques to measure the abundance of many or all gene products such as mRNAs or proteins within a biological sample. • A more focused functional genomics approach might test the function of all variants of one gene and quantify the effects of mutants by using sequencing as a readout of activity. • Together these measurement modalities try to quantitate the various biological processes and improve our understanding of gene and protein functions and interactions.
- 8. Techniques at DNA level • Epistasis is a phenomenon in genetics in which the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes Genetic interactions • Techniques have been developed to identify sites of DNA-protein interactions. These include Chip- sequencing, CUT&RUN sequencing and Calling Cards. DNA/protein interactions • These regions of open chromatin are candidate regulatory regions(that are acessible). These assays include ATAC-seq, DNase-Seq and FAIRE-Seq. DNA accessibility assays
- 9. Techniques at RNA level • Microarrays Microarrays measure the amount of mRNA in a sample that corresponds to a given gene or probe DNA sequence. Probe sequences are immobilized on a solid surface and allowed to hybridize with fluorescently labelled “target” mRNA. The intensity of fluorescence of a spot is proportional to the amount of target sequence that has hybridized to that spot, and therefore to the abundance of that mRNA sequence in the sample
- 10. DISADVANTAGES • gene expression studies were done with hybridization- based microarrays. Issues with microarrays include cross- hybridization , poor quantification of lowly and highly expressed genes, and needing to know the sequence a priori. • Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, next-gen sequencing of cDNA (notably RNA-Seq).
- 11. Serial analysis of gene expression Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags(which are DNA by means of cDNA) that correspond to fragments of those transcripts and they are analyzed
- 12. RNA-Seq RNA-Seq (named as an abbreviation of "RNA sequencing") is a particular technology- based sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome.
- 13. Massively Parallel Reporter Assays (MPRAs) Massively parallel reporter assays is a technology to test the cis-regulatory activity of DNA sequences STARR-seq STARR-seq (short for self-transcribing active regulatory region sequencing) is a method to assay enhancer activity for millions of candidates from arbitrary sources of DNA. It is used to identify the sequences that act as transcriptional enhancers in a direct, quantitative, and genome-wide manner Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation Perturb-seq
- 14. Techniques at protein level yeast two-hybrid system Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
- 15. Affinity chromatography Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between antigen and antibody, enzyme an d substrate, receptor and ligand, or protein and nucleic acid. It is a type of chromatographic laboratory technique used for purifying biological molecules within a mixture by exploiting molecular properties, e.g. protein can be eluted by ligand solution.
- 16. Mass spectrometry Mass spectrometry (MS) is an analytical technique that measures the mass-to- charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures
- 17. Deep Mutational Scanning In Deep mutational scanning every possible amino acid change in a given protein is first synthesized. The activity of each of these protein variants is assayed in parallel using barcodes for each variant. By comparing the activity to the wild- type protein, the effect of each mutation is identified. Loss-of-function techniques Mutagenesis, RNAi, CRISPR screens
- 19. • In molecular biology there are number of techniques are available to understand the function of the gene • For identification of gene function there are two methods used commonly • Forward genetics (Classical genetics) • Reverse genetics Forward genetics • A traditional approach to the study of gene function that begins with a phenotype (a mutant organism) and proceeds to a gene that encodes the phenotype • It depends upon the identification and isolation of random mutation that affect the phenotype of interest
- 20. Reverse genetics • A molecular approach that begins with a genotype ( a DNA sequence) and proceeds to the phenotype by altering the sequence or by inhibiting its expression • It is possible due to the advancement in the molecular genetics
- 21. Forward genetics vs reverse genetics
- 22. Techniques of forward genetics • Initially scientist are depends on the mutation that are occurs naturally, but after the discovery of mutagenic agent increases the rate of mutation • First experimentally created mutation - using X rays - to induce X linked mutation in Drosophila melanogaster- by H. J. Muller in 1927 • Two types of mutations were majorly used in the forward mutation – A. Spontaneous mutation B. Creating random mutation Spontaneous Mutation • It arises spontaneously from natural changes in DNA structure or from error in the replication • i.e. mutation results from both internal and external factors • Where as the changes caused by the radiation or environmental chemicals are called as induced mutation Creating random mutation • It depends upon the identification and isolation of random mutation that affect the phenotype • Radiation (X rays), chemical mutagen (EMS) and transposable elements (insert within a coding region and disrupt the amino acid sequence ) are used to create the mutation
- 23. NON RECOMBINANT RECOMBINANT •Large-scale random mutagenesis and screening •Chemical mutagenesis or TILLING •Gene silencing(RNAi) •Next generation sequencing. •Homologous recombination •Genome edition(ZFNS,TALENS,CRISP ER) Reverse genetics techniques
- 24. Large-scale random mutagenesis and screening • In such methods, cells or organisms are exposed to mutagens such as UV radiation or mutagenic chemicals, and mutants with desired characteristics are then selected. • Hermann Muller discovered in 1927 that X-rays can cause genetic mutations in fruit flies, and went on to use the mutants he created for his studies in genetics. • For Escherichia coli, mutants may be selected first by exposure to UV radiation, then plated onto an agar medium. The colonies formed are then replica-plated, one in a rich medium, another in a minimal medium, and mutants that have specific nutritional requirements can then be identified by their inability to grow in the minimal medium. • instead of screening for a particular phenotype, screen your gene of interest for nucleotide changes • Typically requires that screen 1000’s or 10,000’s of individuals • This is done by performing PCR for gene of interest and looking for slight differences in the migration of the PCR product on a gel or column
- 25. Tilling • Targeting Induced Local Lesions IN Genome (TILLING) • Identifying point mutations in a specific gene by heteroduplex analysis. • Introduced in 2000, using the model plant Arabidopsis thaliana by McCallum. • Identified diverse versions of genes in a germplasm and acquiring information about their gene function. • An expansion of the TILLING technique is Eco TILLING, which can be used to discover point mutations or polymorphisms in Natural populations. • Both can be used to identify unknown and known point mutations from a set of candidate genes.
- 26. Steps involved Seeds are mutagenized by EMS M1 population is grown from mutagenized seeds Resulting M1 plants are self fertilized to produce M2 generation The M2 generation of individuals are used to prepare DNA samples for mutational screening. The DNA samples are pooled eight fold to maximize screening efficiency
- 27. Arrayed on 96-well micro titer plates Equal amounts of DNA from each individual sample. The pooled DNA samples are amplified by PCR using primers, Heteroduplex Formation by PCR amplification at the place of mutation heteroduplex is cleaved at the 3’ end of the mismatch by Endonuclease Cel I. The cleaved products can be separated easily by 10%
- 29. Gene silencing(RNAi) • Long ds-RNA cleaved by an endo- nuclease (Dicer) to form si-RNA. • Once the si-RNA enters the cell it get incorporated into other protein to form RISC ( Argonaut , slicer) • Si-RNA unwound to form single stranded si-RNA. • Si-RNA scan and find a complementary m- RNA • Bind to the targeted m-RNA and cleaved it.
- 30. Next generation sequencing •Technologies developed after that are known as next generation sequencing. •NGS enables the sequencing of biological codes at a very rapid pace with low cost per operation. •This is the primary advantage over conventional methods. •For example Billions of short reads can be sequenced in one operation.
- 31. Homologous recombination • Recombination is the exchange of genetic information between DNA molecules; when the exchange is between homologous DNA molecules it is called homologous recombination • Works in bacteria, yeast, mice and other mammals • Homologous recombination can be used to produce specific mutation in an organism. Vector containing DNA sequence similar to the gene to be modified is introduced to the cell, and by a process of recombination replaces the target gene in the chromosome. This method can be used to introduce a mutation or knock out a gene, for example as used in the production of knockout mice