by Martin Piskacek Overview of molecular biology exp. methods used in pathophysiology Sequencing DNA Sequencing developed by Frederick Sanger • determine the DNA sequence • based on DNA replication • dideoxy nucleotide missing hydroxyl group at 3’ position terminate DNA extention RNA natural nucleoside in DNAtoxic nucleoside analog DNA synthesis and termination • DNA chain extends in 5’3’ direction • Requires nucleotides 3’ –OH group • Extension of the DNA chain is randomly terminated by dideoxynucleotides toxic nucleoside analog DNA Extension by dATP, dGTP, dTTP, dCTP 5’-TACGGATT-3’ 3’-ATGCCTAAGGCTACCGA-5’ size 5’-TACGGATTCCGATGGCT 17 nt 3’-ATGCCTAAGGCTACCGA-5’ 17 nt template DNA is extended 5’3’ primer resulted in to single band acquired on the gel electrophoresis DNA Extension by dATP, dGTP, ddTTP, dCTP 5’-TACGGATT-3’ 3’-ATGCCTAAGGCTACCGA-5’ size 5’-TACGGATTCCGAT 13 nt 3’-ATGCCTAAGGCTACCGA-5’ 17 nt template DNA is extended 5’3’ primer resulted in to two bands acquired on the gel electrophoresis DNA Extension by dATP, ddGTP, dTTP, dCTP 5’-TACGGATT-3’ 3’-ATGCCTAAGGCTACCGA-5’ size 5’-TACGGATTCCG 11 nt 3’-ATGCCTAAGGCTACCGA-5’ template DNA is extended 5’3’ primer resulted in to two bands acquired on the gel electrophoresis Primer: 5’-CGTCGAC-3’ Template : 3’-GCAGCTGATGCCTATGGCTACCGA-5’ Reaction A size CGTCGACTA 9 nt CGTCGACTACGGA 13 nt CGTCGACTACGGATTCCGA 19 nt CGTCGACTACGGATTCCGATGGCTA 25 nt DNA Extension by dATP, dGTP, dTTP, dCTP + small amount of ddATP multiple bands acquired on the gel electrophoresis Dideoxy DNA Sequencing Four separate DNA sequencing reactions are set up Reaction G: dATP, dGTP, dTTP, dCTP + ddGTP Reaction A: dATP, dGTP, dTTP, dCTP + ddATP Reaction T : dATP, dGTP, dTTP, dCTP + ddTTP Reaction C : dATP, dGTP, dTTP, dCTP + ddCTP DNA polymerase is added to each and - complementary strand is synthesized - Dideoxy-nucleotides terminate synthesis DNA fragments analytical separation • Incorporation of a particular ddNTP produces DNA fragments • DNA fragments are then run on a polyacrylamide gel (PAGE) • DNA fragments, which differ by single nucleotide in size can be separated and visualized or detected • Fragments from the four reactions (G, A, T, C) are run apart • Location of a band on the gel corresponds to the position of that nucleotide in the linear sequence Capillary electrophoresis 24 / 48 capillaries high resolution separation technique Fluorescence Automated Sequencing • Each of the dideoxynucleotides is labelled with a different fluorescent dye • All four of the reactions (G, A, C, T) can take place in same tube • The fragments are therefore loaded on to same lane of the gel • fluorimeter and computer are linked to the gel and they detect and record the dye attached to the fragments as they come off the gel • Sequence is determined by the order of the dyes coming off the gel Pyrosequencing Sequencing-by-synthesis (SBS) • ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light • Each bases has a different label on the final phosphates - label of released pyrophosphate is recorded Oxford Nanopore Technologies Ltd • 2048 membrane wells • Motorprotein / Adaptor • DNA or direct RNA seq • 400 bp/s output monitored by current flux about 20-30 pA under 180 mV • Adaptation of α-hemolysin α-HL nanopores for the identification of single molecules (pore-forming toxin from Staphylococcus aureus responsible for the cell lysis) • The leader adapter guides the dsDNA fragments to the vicinity of pores, and the sequencing process begins when the leader motor protein unzips the dsDNA enabling the first strand (template) to pass through the nanopore one base at a time • bacteriophage phi29 DNA polymerase (phi29DNAP) Genomic DNA footprinting - applied seq. dimethyl sulfate (DMS) in vivo protection assays labeling / Sanger seq. gel separation DNAI protection crosslinking (glutaraldehyde) - CHIP (PCR/seq) Methylation-specific PCR (MSP): used to detect methylation of CpG islands in genomic DNA. sodium bisulfite treatement , which converts unmethylated cytosine bases in to uracil sulphonization /de-amination /de-sulph. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation. Deep Sequencing • error versus mutation • cell clones representation • identification of SNV Transcriptomics RNA-seq alternative to Microarray cDNA seq SAGE Serial analysis of gene expression cDNA is generated from the RNA but is then digested into 11 bp "tag" fragments using restriction enzymes CAGE Cap Analysis of Gene Expression biotinylation of the 7-methylguanosine cap of Pol II transcripts, to pull down the 5’-complete cDNAs reversely transcribed from the captured transcripts Key features for abm's RNA-Seq service Sequencing Platform Illumina Sequencing Scale 8 million reads for rapid expression analysis 40 million reads for detecting alternative splice forms 80 million reads for identifying low-abundance coding and noncoding transcripts Higher coverage available up to 400 million reads per sample Starting Material 0.1μg – 4μg of Total RNA 10-100ng of Poly-A enriched mRNA 10-100ng of rRNA depleted RNA Sequencing Type 75 bp single end or paired end sequencing. Longer read lengths available upon custom request. Bioinformatics Analyses FastQC on raw sequencing data (included) Read alignment and estimation of gene expression (included) Differential gene expression analysis Functional annotation Project No. Part Number/ Description Qty. Unit Price (USD) Line Total (USD) 16 human T cell of RNA library prep & sequencing Hiseq, PE150 Q30≥80% 1 Total RNA Isolation 16 30 480 2 RNA library prep ( rRNA depletion by Ribo-ZeroTM& directional library) 16 280 4480 3 Sequencing of PE150 (40M reads=12G/sample) 16 240 3840 4 Data Delivery via 1T hard disk drive 1 150 150 Total USD8950.00 Hi-C all-vs-all high-throughput chromosome conformation capture cross linking + fragment ligation high throughput fragment sequencing Topologically Associating Domains TADs Topologically Associating Domains TADs and chromatin associated RNA Nuclear Pores Bind Inducible Genes Nup98 ChIP-seq Monitoring of nucleosome occupancy in the human genome: ChIP-seq specifically and genome-wide assay chromatin immunoprecipitation with DNA sequencing, to obtain the DNA sequences which interact with transcription factors and nucleosome-associated sequences DNase / MNase-seq chromatin accessibility assay genome-wide DNase I hypersensitive sites sequencing micrococcal nuclease digestion (Staphylococcus aureus) identification of accessible DNA regions in the genome DNA bound to histones or other chromatin-bound proteins transcription factors / repressors / regulators SWI may remain undigested - ATAC-seq THSS - Tn5 hypersensitive site Assay for Transposase-Accessible Chromatin using sequencing mutated hyperactive transposase Tn5 transposase inserts sequencing adapters into unprotected regions - FAIRE-seq Formaldehyde-Assisted Isolation of Regulatory Elements formaldehyde cross-link DNA and proteins, sonication and phenol-chloroform extraction -nucleosomes will preferentially sit in the organic phase and nucleosome-depleted regions will be purified from aqueous phase ChIP-seq Heatmaps and average profiles showing Foxa1, ERa, and AR ChIP-seq signal co-localized around transcription start sites TSS in sperm chromatin at all Refseq annotated genes. Sites are ordered by k-means clustering of RNAPIISer5ph and RNAPIISer2ph signal between TSSs and TTSs