2/5/16. Honeypot Ants. DNA sequencing, Transcriptomics and Genomics. Gene sequence changes? And/or gene expression changes?

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1 2/5/16 DNA sequencing, Transcriptomics and Genomics Honeypot Ants "nequacatl" BY2208, Mani Lecture 3 Gene sequence changes? And/or gene expression changes? gene expression differences DNA sequencing, Transcriptomics and Genomics Allow us to analyze gene sequences and expression levels of all genes in an organisms. In many cases, simultaneously. This allows us to study how this varies in: the animal kingdom, in populations, individuals, in cell types, and in health and disease. How do we do analyze gene expression and sequences?? What have we learned from this so far? What could we learn in the future? 1

2 Key Features of Transcription (making RNA from DNA) ~ 30,000 human genes ~ 150,000 (?) human mrnas - a rough estimate Promoter which controls levels, cell specificity and temporal aspects of transcription gene Genomic DNA DNA Transcription pre-mrna Alternative Splicing mrna Transcription mrna transcript Genome Transcriptome (for protein coding RNAs) Human 30,000 genes 150,000 unique mrnas Alternative splicing of mrnas! Alternative splicing yields different mrnas and protein isoforms Gene Introns = Intervening regions ATG Gene TAA Exons = oding regions Transcription Transcription Nuclear pre-mrna The Processed or mature cytoplasmic mrna Splicing: removal of introns Translation: of mature mrna E1 E2 E4 Alternative Splicing Translation E1 E3 E4 Alternatively spliced mrnas Encoded protein Protein 1 Protein 2 Unique protein isoforms 2

3 ! Measuring the abundance of a specific gene s mrna transcripts Northern blotting can be used to detect specific mrnas Investigating gene expression: 2. Measuring changes in gene expression via mrna 1. Separate total RNA by gel electrophoresis 2. Transfer the separated RNAs out of the gel and onto a nylon membrane (85%) Nylon membrane Agarose gel Electroblotting drives the RNA out of the gel and onto the membrane! Detecting the mrna transcripts of a specific gene (DNA probe hybridization) Hybridize to a labeled DNA of the gene of interest needs to be labelled e.g. with radioactive 32 P Northern Blot analysis of two plant mrnas during flower development mrna bands detected by the Gene1 and Gene2 probes Time (days) Using DNA complimentary to the mrnas (cdna) mrna Gene 1 mrna Gene 2 Gene1 and Gene 2 show developmentally regulated expression during flower development 3

4 ! Reverse transcriptase makes DNA from an RNA template polya tail (almost all mrnas have one)! Measuring the abundance of mrnas via amplification of cdna Reverse Transcription-PR (RT-PR) Add oligo dt primer Add Reverse transcriptase Quantifies mrna indirectly via PR amplification of cdna copies Heart Lung Kidney Liver Brain Reverse transcription Amplified cdna Gene 1: Kinase Amplified cdna Gene 2: Actin Add RNAse H DNA polymerase Microarrays to measure changes in gene expression across the whole genome! Using DNA microarrays for genome-wide analyses of gene expression A 2-D microarray of oligonucleotide probes representing each gene in the genome 30,000 50,000 oligonucleotide probes per microarray (DNA chip) Each probe spot has a defined location in the array Extract RNA from each sample Oligonucleotide probes fixed to chip surface Reverse transcribe RNA to cdna Gene 1 Gene 2 Red label Green label Sample A cdna labelled with Red fluorophore Sample B cdna labelled with Green fluorophore 4

5 ! Microarray hybridization and processing! Microarray example I: How does gene expression differ between normal cells vs. cancer cells? Labelled cdnas cdna hybridization to specific array probes Genes that are upregulated in cancer.cells are potential drug targets Microarray probes The gene expression profile may be diagnostic for particular cancer sub-types and aid diagnosis Data analysis Measurement of fluorescence yield for each gene probe Laser excitation of hybridized cdna Hybridize to microarray Identify gene expression differences etc Microarrays are typically used for sampling expression of relatively small numbers of diagnostic genes. They are also used to screen a panel of diagnostic mutations mrna profiling is now via large scale cdna sequencing (costs falling!). Affymetrix (microarrays) DNA sequencing Illumina (NextGen Sequencing) 5

6 ! Determining the base sequence of a DNA molecule Key to Sanger sequencing is the development of di-deoxynucleotides to terminate chain synthesis (and gels to resolve DNA fragments of similar size) DNA sequencing today generally uses the Sequencing by synthesis method Sequencing by synthesis was developed by Fred Sanger G H H Fred Sanger G Deoxy G PPi (pyrophosphate) Dideoxy G The absence of the 3 -OH group in a di-deoxy nucleotide prevents further elongation of the chain It terminates chain synthesis! General principle of sequencing by synthesis! Fluorescently-labelled dideoxynucleotides made sequencing more efficient The site to which the primer anneals defines the start point Use a radio-labelled primer Add dntps and DNA polymerase to extend the primer Separate the newly synthesized DNA strands by size using gel electrophoresis through a polyacrylamide gel Use autoradiography to reveal the newly-synthesized DNA strands Template strand 3 -GGTATTAAAAGGTGTAAATTAGGGA-5 5 -AGTGGAA Primer T G A Smallest DNA molecules Laser causes bands to fluorescence ombine the 4 reactions Separate fragments on a gel analysis Each dideoxynucleotide is tagged with a unique colored fluorophore Electropherogram 6

7 2/5/16 Pyrophosphate Sequencing Mostafa Ronaghi, Mathias Uhlén and Pål Nyrén* Based on detecting pyrophosphate released during DNA extension Next-Generation Sequencing Very fast, parallel short reads of Millions of (35-400bp) sequences reads. Roche/454 Illumina/Solexa Life Technologies/ABI G Pyrogram (sequencing from a template) PPi (pyrophosphate) Helicos Pacific Biosciences omplete Genomics The first cycle in a round of Solexa sequecing Solexa/illumina uses clusters of single DNA molecule. Shotgun sequencing Your genome sequence for $ Sequence millions of small random fragments (10X coverage). omputers assemble the sequence 7

8 Human Genomics To identify mutations for disease, disease resistance, drug sensitivity, and traits Phenylketonuria! Sequenced genomes.. List_of_sequenced_eukaryotic_genomes Why? R5 delta 32 mutation confers" resistance to Aids" Understand biodiversity, mechanisms, (develop drugs) industrial proteins etc. stephen_friend_the_hunt_for_unexpected_genetic_heroes? language=en Gene Prospecting Sargasso Sea 200 litres of water. Filter (multiple steps) Extract DNA Shot gun sequence Assemble / compare. 1 ml of water as 1 million bacteria, 10 million viruses. Identified ,000 species of bacteria 1.2 million new genes (and doubled the number of protein families known)

9 2/5/16 Synthetic Biology Synthia: the artificial bacterium Mycoplasma mycoides 1,200,000 bp Hamilton O Smith raig Venter Undergraduate Members: Team Ben Wilson, Patrick King, David Fallon, Arnas Petraukas, Daire Gannon, Emma ooke, Remsha Afzal, Zaneta Najda, Marlena Mucha, Anya Aleshko. Apr 25, 2015 Nov 01, 2015 Trinity Undergraduates Aim to Build AntiMalarial 'Biobrick.' Members of the Genetics Department amongst winners of International Genetically Engineered Machine (igem) competition. Trinity ollege Dublin igem 2015 Project Introduction 9