FunctionalgenomicsFunctional genomics is a branch of genomics that focuses onpatterns of gene expression and interaction in the genome.
especially, it isused to measure the expression levels of RNA and proteins to increaseunderstanding of the biological function of the genes of a cell. Functional genomics focuses on the dynamic aspects such as genetranscription, translation, and protein–protein interactions. Functional genomicsattempts to answer questions about the function of DNA at the levels of genes,RNA transcripts, and protein productsObjective:The main aim of functional genomics is to utilize thewealth of data produced by genomic and transcriptomic projects to describe thefunctions and interactions of a genome and phenotype. It looks at the dynamicaspects of the genes, such as transcription, translation, expression, andprotein-protein interactions, and aims to explain these processes based oninformation about the genome.
Functional genomics promises to expand thepractical knowledge of the effect that an organism’s genome has on itsbiological function. This could have a significant impact on the way we treathuman genetic diseases in the future and has the potential to revolutionize ourcurrent health system.Techniques:• Transfection• Real-time PCR• RNA interference• Mutational analysis• SNP analysis• Microarray analysisMicroarray:Definition: It is a 2D array ona solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughputscreening .It is miniaturized,multiplexed and parallel processing and detection methods.
Probes that contain thousands of spottedsamples are immobilized on a solid support (a microscope glass slides orsilicon chips). The spots can be DNA, or oligonucleotides or cDNA. These areused to conclude complementary binding of the unknown sequences thus permittingparallel analysis for gene expression and gene discovery. An experiment with asingle DNA chip can give information on thousands of genes instantaneously.
Theproper arrangement of the probes on the support is essential as the location ofeach spot on the array is used for the identification of a geneMicroarrayPrinciplemRNA is an intermediate moleculewhich transfers the genetic information from the cell nucleus to the cytoplasmfor the synthesis of proteins. Many copies of mRNA parallel to the certaingenes are produced by a process termed as transcription. These mRNAs producethe corresponding protein by a process known as translation.
This helps in theconsiderate of various processes behind every different genetic expression.mRNA degradation occurs easily, it is necessary to convert it into a morestable cDNA form. Labeling of cDNA is done by fluorochrome dyes Cy3 (green) andCy5 (red). The principle after microarrays is that complementary sequences willbind to each other. By using restriction endonucleases, theunknown DNA molecules are cut into fragments. then fluorescent markers areattached to these DNA fragments.
These are then permitted to react with probesof the DNA chip. Then the target DNA fragments along with complementarysequences bind to the DNA probes. The remaining DNA fragments are washed away.The target DNA pieces can be identified by their fluorescence emission bypassing a laser beam. A computer is used to record the pattern of fluorescenceemission and DNA identification. This technique of employing DNA chips is veryrapid, besides being sensitive and specific for the identification of severalDNA fragments simultaneously.Types of MicroarraysDepending upon the kind of immobilized sample, the Microarrayexperiments can be characterized in three ways: Microarray Expression Analysis: In this experimentalsystem, the cDNA resulting from the mRNA of known genes is immobilized.
Thesample has genes containing both the normal as well as the diseased tissues.Spots with more intensity are found for diseased tissue gene if the gene isover expressed. Then we compare the expression pattern of a gene for a diseasewith a normal one. Microarray for Mutation Analysis: For this analysis,the researchers use gDNA. The genes differ from each other by a singlenucleotide base and known as SNP. Comparative Genomic Hybridization: It is used for theidentification in the increase or decrease of the vital chromosomal fragmentshiding genes involved in a disease.Applications of MicroarraysGene Discovery: DNA Microarray technology aids in theidentification of new genes, know about their operative and expression levelsunder different conditions.Disease Diagnosis: DNA Microarray technology benefits researcherslearn about different diseases such as heart diseases, mental illness,infectious disease and particularly the study of cancer.
Until recently,different types of cancer have been classified.Transfection:It is the process of intentionally introducing naked orpurified nucleic acids into eukaryotic cells.Cells that have incorporated foreign DNA are called transfectants.Transient transfectionInthis foreign gene do not integrate it into their genome. Thus, the new genewill not be replicated. These cells express the transfected gene for a finiteperiod, usually several days, after which the foreign gene is lost through celldivision or other factors.Stable transfection In a small proportion of transfected cells,the foreign gene is integrated into the cells’ genome.
The symbol of stablytransfected cells is that the foreign gene becomes part of the genome and istherefore replicatedFactors that affecting transfection:These are the factors that depend on the transfection.Ø CelltypeØ Cellhealth and viabilityØ MediaØ Typeof molecule transfectedTransfection Methods:Calcium phosphate:Calciumphosphate co-precipitation is extensively used because the components areeasily obtainable and reasonable in price. Another advantage is itsapplicability to produce stably-transfected cell lines, allowing for long-termgene expression studiesElectroporation Electroporation is a commonly used physicalgene transfer method. Principle – Cells and DNA are suspended in anelectroporation buffer – High voltage of electricity are applied to the cells –Electrical pulse creates a potential difference across the membrane, inducestemporary pores in the cell membrane for DNA entry.
RNAinterference:RNAi is a process in biology that usually inhibits theexpression of genes related to or any orther protein translation mechanisms.Other names used for it in previous times are PTGS (post transcriptional genesilencing), quelling and co-suppression. DiscoveryThe process of PTGS was first discovered in plants as the fightagainst foreign RNA or DNA as the part of defense machinery. It is used toregulate the expression of endogenous genes.Historyof RNAi Need of interferenceo DefenseMechanismo Genomewide regulationCharacteristics ofRNAi · Theparent sequence of targeted mRNA is lost after RNAi· AfterRNAi the dsRNA and ssRNA cannot work· Andthe dsRNA should not be directed to intron but exon so that it can workeffectively· Andthe homology of targeted mRNA and dsRNA is essentialUses of RNAiØ Gene RedundancyØ Testing Hypotheses of Gene Function Target ValidationØ Studying cell divisionØ Functional screeningØ Pathway AnalysisMechanism of RNAi Importanceof RNAiØ Early identification of function of geneØ Identification of unknown sequence ofthe geneØ Agricultural importanceØ In different diseases down regulation ofmany mutated and harmful genesØ Knock out of genes Ø siRNA use to determine human genes RNAi and plant technology· to lower plants toxins bystable and heredity phenotype of RNAi in stocks of plants· produce the stocks of cottonwhich have lower level of delta-cadinene synthase· reduction in gossypolproduction in cotton plants for safety from pests· lower the level of allergensin tomato plants· reduce the carcinogenicityof plants like tomato and cassava plants· production of opium poppy· resistance to pathogens andviruses· insertion of dietaryantioxidants in plants e.g. tomatoes for fortificationClinical Applications of RNAi· stem cell biology· oncology· hepatitis C· macular degeneration· infectious diseases· hematology· virus targeting· respiratory infections· gene silencing in HIVReal Time PCRIt is basedon the polymerase chain reaction and known as quantitative PCR for theamplification of target molecule mostly the DNA molecule for differentpurposes. History:Working PrincipleTounderstand the working principle of real time PCR the plot is shown in which onthe x axis the number of PCR cycle and on y axis the fluorescence is shown ofthe amplified product.
Fig. Amplification plot in whichbaseline subtracted fluorescence vs. no. of PCR cyclesIn exponential phasethe product of PCR doubles after every cycle and the components of the reactionare consumed and then become limited and after that the very samereactionenters in the non-exponential plateau phase.Not initially but afterthe rate at which the product becomes detectable the fluorescence signal isshown. Cq value is the cycle numberat the exponential phase when the reactants are unlimited in real time PCR. Itis determined as the template present in amplification reaction.
As the smallamount of template, more amplification for the fluorescence signals to beobserved and thus have high Cq.RealTime PCR procedureThe fluorescenceemitted is the most basic component of the real time PCR as the reportermolecule increases its amplification after each cycle and this is categorizedin two types:1. DNA Binding Dyes (Non-specificdetection)2.
Target Specific Probes (Specificdetection)DNABinding DyesThese dyes are used asthe fluorescence reporters in the reaction. It increases as the amplificationof the product increases. And the graph of fluorescence vs. amplification showsthe relationship between them. The dye use mostly id SYBER Green as it bind inthe minor groves of the DNA in double stranded structure. Ethidium Bromide canalso be used but due to its carcinogenic nature it is avoided.
These dyes arecheap and simple in PCR reaction and the drawback is that it causes productaccumulation sometimes.TargetSpecific ProbesThe oligonucleotideslabeled probes are used for the specific detection. Following are the exampleof some probes:Ø TaqManprobeØ ScorpionprimersØ MolecularBeaconsØ FRETHybridization probe Applications of Real Time PCR · In viral or genomic DNA the measurementof copy number· Efficiency of Drug therapy· Measurement of the DNA damage· Microarray results verification· Expression studies of quantitative mRNA· SNP genotyping· Allelic discrimination assaysRealTime PCR vs. Traditional PCRIn real time PCR theearly phase detection takes place as compared to the traditional PCR and thetraditional methods use conventional techniques like gel electrophoresis todetect amplification of the product after the reaction. FuturePerspectives, Challenges and Progress in Functional Genomics:Soon there are many different research avenues that wouldsignificantly benefit from the application of this platform.Fig. The overall flow and hierarchy of functionalgenomics and systems biologyFunctional foods:The development and design of functional foods could beenhanced by the application of functional genomics tools to identify beneficialpathways and engineer them into or out of specific plants and crops.
This wouldresult in the enhancement of food quality without the need to modify foods atthe postharvest stage.Genome sequencing and functional genomics:The analysis ofentire ecosystems and the potential for their management and modification woulddramatically benefit from the lower cost of DNA/RNA sequencing. Projects thatinvolve the sequencing of 100s or even 1000s of individual plantsgenomes/transcriptomes from an ecosystem would be possible, preciselydetermining the genetic variability of the system. When other species will beincluded in this genome/transcriptome analysis, interactions between differentspecies could be traced to specific genes and pathways, and new and novelinteractions could be identified. Of course, the health of the ecosystem andits response to global climatic changes could be determined as well at thegenomics level.
Fig. Genome sequencing and functional genomicsMedicinal discovery:Medicinal discoveryis perhaps one of the most important applications for functional genomics.Combined with advanced tools to detect compounds with a medicinal property,functional genomics could identify the precise pathways and genes involved inits biosynthesis. These could then be used for drug production under controlledconditions and the development of novel drugs with different properties.Biofuel development:Biofuel developmentis a hot topic these days and functional genomics is already applied toidentify and engineer different pathways into plants and algae.
The massivesequencing of different plant and algae genomes/transcriptomes, as well as thedifferent metagenomics projects under way are likely to significantly enhancethese efforts identifying, characterizing and engineering new sources forbiofuels, some could even be from unexpected sources, or produced by proteinswhich currently have an unknown function.Functional Genomic in Plant Research:Availability of vast genomic data has resulted in itswide applicative prospects in exploring the genes involved in differentbiosynthetic pathways. Some emerging functional genomic tools are showingpotential in the field of plant research based on secondary data and haveresulted in better utilization of natural medicinal resources. From the lastfew years, the focus is on the understanding of molecular basis of plant responseto different environmental stresses.
Several genes involved in stress responseshave been studied using functional genomic approaches. The recent availabilityof next-generation sequencing and genome-editing tools provides sufficientopportunity to introduce targeted modifications in the genome competently.These new approaches further reveal the functional aspects of variouscomponents of the genome in various plants and offer potential opportunitiesfor designing the plants as per our choice.Recent research in Cotton FunctionalGenomics:Functionalgenomics has transformed from futuristic concept to well established scientificdiscipline during last decade. Cotton functional genomics promise to enhancethe understanding of fundamental plant biology to systematically exploitgenetic resources for improvement of cotton fiber quality and yield, as well asutilization of genetic information for germplasm improvement.
Conclusion:The recentdevelopment in genomics data joined with functional genomics, bioinformaticsand systems biology is going to dominant. The future of scientific researcharises a the big question of that what lies ahead beyond these development.