Functional Genomics Unit II - PowerPoint Presentation

1. Functional GenomicsUnit II

2. IntroductionGenomics – It is the study of genomes.The field of genomics comprises of two main areas:Structural genomicsFunctional genomics

3. Structural genomicsIt deals with genome structures with a focus on the study of genome mapping and assembly as well as genome annotation and comparison.

4. Functional genomicsIt is largely experiment based with a focus on gene functions at the whole genome level using high throughput approaches.Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures.

5. Techniques and applicationsFunctional genomics includes function-related aspects of the genome itself such as mutation and polymorphism (such as single nucleotide polymorphism (SNP) analysis), as well as measurement of molecular activities. It also comprise a number of "-omics" such as transcriptomics (gene expression), proteomics (protein production), and metabolomics.

6. 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.

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8. Genetic Interaction MappingSystematic pairwise deletion of genes or inhibition of gene expression can be used to identify genes with related function, even if they do not interact physically. Epistasis refers to genetic interactions in which the mutation of one gene masks the phenotypic effects of a mutation at another locus.Systematic analysis of these epistatic interactions can provide insight into the structure and function of genetic pathways.

9. EpistasisEpistasis refers to the fact that effects for two different gene knockouts may not be additive; that is, the phenotype that results when two genes are inhibited may be different from the sum of the effects of single knockouts.Epistasis has a large influence on the shape of evolutionary landscapes, which leads to profound consequences for evolution and evolvability of phenotypic traits.

10. Systematic analysis of these epistatic interactions can provide insight into the structure and function of genetic pathways. Examining the phenotypes resulting from pairs of mutations helps in understanding how the function of these genes intersects. Genetic interactions are generally classified as either Positive/Alleviating or Negative/ Aggravating.

11. Fitness epistasis (an interaction between non-allelic genes) is positive, when a loss of function mutation of two given genes results in exceeding the fitness predicted from individual effects of deleterious mutations, and it is negative (aggravating) when it decreases fitness.

12. Detection MethodsHigh-throughput methods of analyzing these types of interactions have been useful in expanding our knowledge of genetic interactions.Synthetic genetic arrays (SGA), Diploid based synthetic lethality analysis on microarrays (dSLAM), and Epistatic miniarray profiles (E-MAP)

13. AdvantagesThis systematic approach to studying epistasis on a genome wide scale has significant implications for functional genomics and mapping of genetic interactions.By identifying the negative and positive interactions between an unknown gene and a set genes within a known pathway, these methods can elucidate the function of previously uncharacterized genes within the context of a metabolic or developmental pathway.

14. Genetic interactions revealed by conditional mapping. Genome plot generated using circos software

15. Transcriptome analysisTranscriptome analysis can be conducted by two approaches:Sequence based approachesMicroarray based approaches

16. Sequence based approachesExpressed sequence tags: ESTs are short sequences of cDNA typically 200-400 nucleotides in length.Obtained from either 5’end or 3’end of cDNA inserts of cDNA library.

17. Advantages of ESTProvide a rough estimate of genes that are actively expressed in a genome under a particular physiological condition.Help in discovering new genes, due to random sequencing of cDNA clones.EST libraries can be easily generated.

18. Drawbacks Automatically generated without verification thus contain high error rates.It is always contaminated by vector sequence, introns, ribosomal RNA, mitochondrial RNA.Weakly expressed genes are hardly found in EST sequencing survey.ESTs represent only partial sequences of genes.

19. SAGE(Serial Analysis of Gene Expression)

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26. ProtocolThe mRNA of an input sample (e.g. a tumour) is isolated and a reverse transcriptase and biotinylated primers are used to synthesize cDNA from mRNA.The cDNA is bound to Streptavidin beads via interaction with the biotin attached to the primers, and is then cleaved using a restriction endonuclease called an anchoring enzyme (AE). The location of the cleavage site and thus the length of the remaining cDNA bound to the bead will vary for each individual cDNA (mRNA).

27. The cleaved cDNA downstream from the cleavage site is then discarded, and the remaining immobile cDNA fragments upstream from cleavage sites are divided in half and exposed to one of two adapter oligonucleotides (A or B) containing several components in the following order upstream from the attachment site: 1) Sticky ends with the AE cut site to allow for attachment to cleaved cDNA; 2) A recognition site for a restriction endonuclease known as the tagging enzyme (TE), which cuts about 15 nucleotides downstream of its recognition site (within the original cDNA/mRNA sequence); 3) A short primer sequence unique to either adapter A or B, which will later be used for further amplification via PCR.

28. After adapter ligation, cDNA are cleaved using TE to remove them from the beads, leaving only a short "tag" of about 11 nucleotides of original cDNA (15 nucleotides minus the 4 corresponding to the AE recognition site).The cleaved cDNA tags are then repaired with DNA polymerase to produce blunt end cDNA fragments

29. These cDNA tag fragments (with adapter primers and AE and TE recognition sites attached) are ligated, sandwiching the two tag sequences together, and flanking adapters A and B at either end. These new constructs, called ditags, are then PCR amplified using anchor A and B specific primers.The ditags are then cleaved using the original AE, and allowed to link together with other ditags, which will be ligated to create a cDNA concatemer with each ditag being separated by the AE recognition site.

30. These concatemers are then transformed into bacteria for amplification through bacterial replication.The cDNA concatemers can then be isolated and sequenced using modern high-throughput DNA sequencers, and these sequences can be analysed with computer programs which quantify the recurrence of individual tags.

31. AnalysisThe output of SAGE is a list of short sequence tags and the number of times it is observed. Using sequence databases a researcher can usually determine, from which original mRNA (and therefore which gene) the tag was extracted.Statistical methods can be applied to tag and count lists from different samples in order to determine which genes are more highly expressed. For example, a normal tissue sample can be compared against a corresponding tumor to determine which genes tend to be more (or less) active.

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47. Microarray

48. Summary of DNA microarray

49. A DNA microarray (also commonly known as DNAchip or biochip) is a collection of microscopic DNA spots attached to a solid surface.DNA microarrays is used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome

50. A typical microarray experiment involves the hybridization of an mRNA molecule to the DNA template from which it is originated.Many DNA samples are used to construct an array. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions.The amount of mRNA bound to each site on the array indicates the expression level of the various genes. All the data is collected and a profile is generated for gene expression in the cell.

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52. Animationshttps://www.youtube.com/watch?v=Pz_yucMksTchttps://www.youtube.com/watch?v=mYWVi7B-13chttp://sumanasinc.com/webcontent/animations/content/dnachips.html

53. ChIP Assay

54. IntroductionChromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome of a particular protein of interest.It gives a picture of the protein-DNA interactions that occur inside the nucleus of living cells or tissues.ChIP is used to determine whether a transcription factor interacts with a candidate target gene and is used with equal frequency to monitor the presence of histones with post-translational modifications at specific genomic locations

55. The first chromatin immunoprecipitation (ChIP) assay was developed by Gilmur and Lis (1984-1986) as a technique for monitoring the association of RNA polymerase II with transcribed and poised genes in Escherichia coli and Drosophila

56. PrincipleDNA and associated proteins on chromatin in living cells or tissues are cross-linked (this step is omitted in Native ChIP).The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion.Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody.The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.

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58. ApplicationChromatin immunoprecipitation (ChIP) assays identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor–DNA binding interactions. The strength of ChIP assays is their ability to capture a snapshot of specific protein–DNA interactions occurring in a system and to quantitate the interactions using quantitative polymerase chain reaction (qPCR). Chromatin IP experiments require a variety of proteomics and molecular biology methods including crosslinking, cell lysis (protein–DNA extraction), nucleic acid shearing, antibody-based immunoprecipitation, DNA sample clean-up and PCR. Additional techniques such as gel electrophoresis are usually used during optimization experiments to validate specific steps.

59. Animationhttps://www.youtube.com/watch?v=4oFdS9EN9Pk

60. Massively Parallel Signature Sequencing (MPSS)MPSS is a open-ended platform that analysis the level of gene expression in a sample by counting the number of individual mRNA molecules produced by each gene.It is developed by Sydney Brenner which is bacteria-free bead based library preparation, “Megaclone” technologyIn MPSS, mRNA transcripts did not need to be known and could be discovered de novoAll clones in a microbead library can be sequenced simultaneously (so, called “massively parallel”)

61. MPSS produces data in a digital format. MPSS Captures data by counting virtually all mRNA molecules in a tissue or cell sample. All genes are analysed simultaneously, and bioinformatics tools are used to sort out the number of mRNAs from each gene relative to the total number of molecules in the sample. Even genes that are expressed at low levels can be quantified with high accuracy. Counting mRNAs with MPSS is based on the ability to identify uniquely every mRNA in a sample by generating a 17-base sequence for each mRNA at a specific site upstream from its poly (A) tail. This 17 base sequence is used as mRNA identification signature. To measure the level of expression of any given gene, the total number of signatures for that gene’s mRNA is counted.

62. PrincipleA sample’s mRNA are first converted to cDNA using reverse transcriptase, which are fused to a small oligonucleotide "tag" which allows the cDNA to be PCR amplified and then coupled to microbeads. After several rounds of sequence determination, using hybridization of fluorescent labelled probes, a sequence signature of ~16-20 bp is determined from each bead. Fluorescent imaging captures the signal from all of the beads, so DNA sequences are determined from all the beads in parallel, approximately 1,000,000 sequence reads are obtained per experiment.

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64. The massively parallel signature sequencing (MPSS) and cDNA tagging strategy.MPSS comprises two modules. First, transcript signatures are extracted from cDNA templates (in a similar way to the serial analysis of gene expression method of tagging) and cloned into a vector flanked by unique oligonucleotide barcode tag sequences. The resulting signature–barcode clones are amplified by PCR and loaded onto microbeads that contain anti-tag barcode sequences. The signature library beads are packaged as a planar array in a flow cell where enzymatic reagents can be pumped through for cycling reactions.

65. Each flow cell can hold up to one million beads representing the same number of cDNA clones. On the beads, adaptors with single-stranded 4 base-pair (bp) recognition sequences, encoding all possible sequence combinations are hybridized to the open-tip of cDNA templates. Only adaptors that perfectly match the open-end of the signatures are ligated to the cDNA molecules on the beads. After washing away unbound adaptors, specific decode probes with fluorescent labels are added sequentially to the flow cell to identify the adaptors on the beads, thereby revealing the 4 bp sequence signatures. The next interrogating cycle is started with the digestion of BbvI (a type-II endonuclease), which binds to the adaptor sequence and cleaves the cDNA, thereby opening the next new 4 bp sequence.

66. ProcedureMPSS signature for mRNAs in a sample are generated by sequencing ds cDNA fragments cloned onto microbeads using the Lynx Megaclone technology

67. Poly (A) mRNA molecules are converted into double-stranded cDNA molecules using biotynalated oligo dT primer. Streptavidin is use to purify biotynalated cDNA. cDNA digested with DpnII. cDNA fragments cloned into a specially designed plasmid vector containing a unique barcode tag. Total 16.8 x 106 million different 32-base sequences available in the reference tag library, and each cDNA clone contains a different sequence. The library of cDNA inserts with oligonucleotide tags are PCR- amplified. The resulting linear molecules are partially treated with an exonuclease to make the 32-base tag single stranded.

68. The 32-base tags at the end of each of the cDNA molecules are hybridised to 32-base complementary tags of microbeads. The end-product is a microbead with approximately 100,000 identical cDNA molecules covalently attached to the surface.

69. Adaptor LigatingEncoded adaptors are ligated to the ends of the cDNA molecules attached to the microbeads.

70. 17 bases signature determinationThe encoded adaptor from the first round is then removed by digestion with Bbv I, which exposes the next four nucleotides as a four-base single- stranded overhang. The process is repeated several times in order to generate a total of 17 bases of sequence

71. SequencingApproximately one million microbeads are loaded into a specially designed flow-cell in a way that allows them to stack together along channels and form a tightly packed monolayer in the flow-cell. The flow-cell is connected to a computer-controlled microfluidics network that delivers different reagents for the sequencing reactions.

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73. A high-resolution CCD camera is positioned directly over the flow-cell in order to capture fluorescent images from the microbeads at specific stages of the sequencing reactions. MPSS system

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75. MPSS has the advantage that it provides in-depth quantitation of virtually all genes that are expressed in a sample. Since there is no requirement for prior knowledge of any gene or genome, it is possible to generate quantitative gene expression datasets from any organism. MPSS dataset involves one million or more signature sequences, it has the sensitivity to quantitate accurately genes that are expressed at very low levels within a cell. No other single technology has these performance characteristics.

76. The advantage of microarray is the high throughput analysis of multiple samples. The microarray and MPSS technologies as being complementary in nature different tools for different types of experiments. e.g. To generate in-depth and quantitative gene expression data for building complex relational databases, MPSS may be the technology of choice. After these databases are mined for interesting biological information, it may be necessary to test whether sets of genes are differentially expressed in a large number of samples (eg tumours of a specific type). Here, the microarray platform be the technology of choice. Both MPSS and at least one of the microarray technologies would seem to be ideal for most investigators

77. RNAi

78. RNA silencingSeveral terms are used to described RNA silencingThey are all phenotypically different but mechanistically similar phenomenaCosuppression or post-transcriptional gene silencing (PTGS) in plantsQuelling in fungiRNA interference in animal kingdom

79. RNA interferenceIt is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules at the stage of translation or by hindering the transcription of specific genes.RNAi targets include RNA from viruses and transposons.

80. Need for InterferenceDefence MechanismsDefence against infection by viruses, etc.,As a defence mechanism to protect against transposons and other insertional elementsGenome Wide RegulationRNAi plays a role regulating development and genome maintenance30% of human genome regulated

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82. MechanismIn InterferenceRNAsiRNA: dsRNA 21-22 ntmiRNA: ssRNA 19-25 nt. Encoded by non protein coding genomeRISCRNA induced silencing complex, that cleaves mRNAEnzymesDicer: produces 20-21 nt cleavages that initiate RNAiDrosha: cleaves base hairpin in to form pre miRNA; which is later processed by Dicer

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94. iRNA - Animationhttps://www.youtube.com/watch?v=cK-OGB1_ELE

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102. RNA interference screensRNA interference (RNAi) screens (repression of individual proteins between transcription and translation) are one method that can be utilized in the process of providing signs to the protein-protein interactions. Individual proteins are repressed and the resulting phenotypes are analysed. A correlating phenotypic relationship (i.e. where the inhibition of either of two proteins results in the same phenotype) indicates a positive, or activating relationship.

103. Phenotypes that do not correlate (i.e. where the inhibition of either of two proteins results in two different phenotypes) indicate a negative or inactivating relationship. If protein A is dependent on protein B for activation then the inhibition of either protein A or B will result in a cell losing the service that is provided by protein A and the phenotypes will be the same for the inhibition of either A or B.

104. If, however, protein A is inactivated by protein B then the phenotypes will differ depending on which protein is inhibited (inhibit protein B and it can no longer inactivate protein A leaving A active however inactivate A and there is nothing for B to activate since A is inactive and the phenotype changes).Multiple RNAi screens need to be performed in order to reliably appoint a sign to a given protein-protein interaction.

105. Mutagenesis

106. IntroductionIn molecular biology and genetics, mutations are changes in a genomic sequence.Mutations are caused by radiation, viruses, transposes and mutagenic chemicals, as well as errors that occur during meiosis or DNA replications

107. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. In nature mutagenesis can lead to cancer and various heritable diseases, but it is also a driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.

108. Mammalian nuclear DNA may sustain more than 60,000 damage episodes per cell per day, as listed with references in DNA damage (naturally occurring).In the laboratory, however, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel functions, as well as mutant strains with useful properties. Initially, the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, but later techniques were developed to introduce specific mutations.

109. TypesSpontaneous hydrolysisModification of basesDNA damage and spontaneous mutationCrosslinkingDimerizationIntercalation between basesBackbone damageInsertional mutagenesisEffects on replication and DNA repair

110. Mutagenesis as a laboratory techniqueMutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, strains of bacteria, or other genetically modified organismsVarious constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detailThe mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.

111. Large number of methods for achieving mutagenesis have been developed. Initially, the kind of mutations artificially induced in laboratory were entirely random, later methods for more specific site-directed mutagenesis were introduced. Since 2013, development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing or mutagenesis of the genome in vivo

112. Random mutagenesisSite-directed mutagenesisCombinatorial mutagenesisInsertional mutagenesisHomologous recombinationGene synthesis