Genomics(functional genomics)

description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it

1. genomics D.INDRAJA

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

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  and  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

18. 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   

19. 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  

20. Forward genetics vs reverse genetics

21. 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

22. 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,CRISPER) Reverse genetics techniques

23. 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  

24. 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 .

25. 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

26. 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% polyacrylamide gel followed by staining with SYBR Green.

27. 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.

28. 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.

29. 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