Functional Genomics Techniques

What are techniques that functional genomics uses?

DNA microarrays, RNA-seq, proteomics, epigenomics, molecular interaction mapping

What are key clues to function?

knowing where and when a protein is expressed and localized and what it interacts with in vivo

What is a transcriptome?

set of all mRNA molecules produced from a genome

What can transcriptome apply to?

complete set of transcripts for a given organism, specific subset of transcripts present in a particular cell type or under specific growth conditions

What is the first reason transcriptome vary?

stimuli yield responses

What is the second reason transcriptome vary?

developmental stages require changes in gene expression

What is the third reason transcriptome vary?

tissues need different proteins for functionW

What is the fourth reason transcriptome vary?

disease states are associated with deviation from healthy state

What is expression profiling?

determining where and when particular genes are expressed

What can mutating one gene alter?

the expression of others

What can genes be used as markers to define?

particular cellular states or cell types

What are the transcriptome profiling techniques?

DNA microarrays, RNA-seq

What is DNA microarray?

hybridization-based approach, incubate fluorescently labelled cDNA with DNA microarrays, intensity fluorescent signal reflects abundancy of RNAs

What is RNA-seq?

sequenced-based approach, directly determine the cDNAs, read counts directly reflect abundancy of RNAs

What does each transcriptome profiling approach monitor?

the steady state levels of transcripts of each genes, combination of synthesis and turnover

What are DNA microarray used to estimate?

the relative levels of gene expression of each gene in a genome in a single experiment

What is DNA microarray primarily used for?

to compare the expression of many genes under different conditions

What is the level of gene transcription proportional to in DNA microarray?

intensity of the fluorescence signal

What is step 1 of DNA microarrays?

isolate mRNAs from cells at two stages of development, each mRNA sample represents all the genes expressed in the cells at that stage

How is mRNA selected in DNA microarrays?

purifying from total RNA using oligoT beads that bind polyA tails of mRNA or rRNA depletion

What is step 2 of DNA microarrays?

convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled dNTPs

What does step 2 of DNA microarray require?

DNA primer, oligo dT to hybridize with mRNA 3’ polyA tail

What is step 3 of DNA microarrays?

add the cDNAs to a microarray, fluorescent cDNAs anneal to complementary sequences on the microarray

What is step 4 of DNA microarray?

each fluorescent spot represents a gene expressed in the cells

What are the fluorescent signals in DNA microarrays obtained by?

laser excitation and array scanning

What does RNA-seq provide?

global info on RNA expression patterns, relative abundance, 5’ and 3’ ends, splicing patterns

What are cDNAs sequence using in RNA-seq?

next generation DNA sequencing technologies and reads are mapped to reference genome

What is step one of RNA-seq?

isolate and purify input RNA

What is step 2 of RNA-seq?

convert the RNA to cDNA and add sequencing adapters

What is step 3 of RNA-seq?

sequence cDNAs using one of the available NGS platforms

What is step 4 of RNA-seq?

analyze the resulting short-red sequences

What do sequence read genome alignments and counts reveal in RNA seq?

sites and levels of gene expression

What NGS technology is commonly used in RNA-seq?

Illumina

What is step 1 of Illumina?

fragmented RNA converted to cDNA, ligate adapters to both ends of cDNA fragments

What is step 2 of Illumina sequencing?

attach DNA to surface

What is step 3 of Illumina?

bridge amplification

What is step 4 of Illumina?

completion of amplification

What is step 5 of Illumina?

sequencing-by-sythesis

What are the materials in Illumina sequencing-by-synthesis?

4 fluorescently labeled nucleotides, primers, polymerase

What is step 1 of Illumina sequencing by synthesis?

take image of first cycle

What is step 2 of Illumina sequencing by synthesis?

remove fluorophore

What is step 3 of Illumina sequencing by synthesis?

remove block on 3’ terminus

What can RNA-seq be used to characterize?

novel transcripts and splicing variants as well as to profile the expression levels of known transcripts

Why does RNA-seq has higher resolution than whole genome tiling array analysis?

mRNA can achieve single-based resolution, where the resolution of microarray depends on the density of probes

What is the proteomes?

set of all proteins produced under a given set of conditions

What can the proteome term be applied to?

complete set of proteins, specific subset of proteins present in a particular cell type or under specific growth conditions

What does proteomics analysis on many samples use?

2D-electrophoresis and mass spectrometry

What is step 1 of 2D-gel electrophoresis?

separate proteins on basis of net charge

What is step 2 of 2D gel electrophoresis?

separate proteins in basis of molecular weight

How many proteins can be resolves using 2D gel electrophoresis?

>1000

What does digestion with trypsin in mass spectrometry do?

give fragments with unique set of sizes

What does mass spec allow?

identification of unknown protiens

What is shotgun proteomics?

identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spec to give protein sequences

What are the differences in transcriptomic and proteomic practical application?

transcriptomics is robust cost-effective and user-friendly, proteomics had problems with purification and stability of proteins

What do you use for identify components of protein complexes?

immuno or affinity purification of protein complexes

What do you use for identifying specific DNA/protein interactions?

ChIp-seq

What do you use to identify specific RNA/protein interactions?

CLIP-seq

What are the reagents for mapping molecular interactions?

antibodies specific to the protein of interest

What is step 1 of getting antibodies?

immunize mouse with antigen

What is step 2 of getting antibodies?

fuse mouse B cells with myeloma cells to produce hybridoma cells

What is step 3 of getting antibodies?

select hybridomas, plate on HAT media to kill off unfused cells

What is step 4 of getting antibodies?

clone hybridoma lines

What is step 5 of getting antibodies?

harvest, purify, and use antibodies

What is co-immunoprecipitation?

immunopurify protein of interest and any associated components, identify co-isolated proteins using mass spec sequencing approaches

What is step 1 of immuno or affinity purification of protein complexes?

purify via immunoprecipitation or selection of tagged protein

What are common protein tags/affinity method?

epitope tags, polyhistidine/nickel chromatography

What is step 2 of immuno or affinity purification of protein complexes?

separate tagged protein and co-purifying protein complexes on SDS gel

What is step 3 of immuno or affinity purification of protein complexes?

excise individual protein bands

What is step 4 of immuno or affinity purification of protein complexes?

digest with trypsin

What is step 5 of immuno or affinity purification of protein complexes?

identify the sequence proteins by liquid chromatography followed by mass mass spec

What is step 6 of immuno or affinity purification of protein complexes?

data base analysis identify proteins from sequences of the peptides

What is ChIP Seq?

combines chromatin immunoprecipitation and high throughput DNA sequencing to identify specific protein/DNA interactions in vivo and genome wide

What can ChIP seq be used for?

TFs, histones, RNA polymerase, DNA repair proteins

What does ChIP seq require?

an antibody that recognizes the protein of interest

What is done to DNA fragment bound to protein of interest in ChIP-seq?

high throughput sequencing

What does CLIP stand for?

crosslinking immunoprecipitation

What is CLIP-seq?

method to identify the RNAs that a particular RNA binding protein interacts with in vivo

What is step 1 of CLIP-seq?

UV irradiate cells

What is step 2 of CLIP-seq?

partial RNase digestion

What is step 3 of CLIP-seq?

purify RNP

What is step 4 of CLIP-seq?

proteinase K/DNase

What is step 5 of CLIP-seq?

ligate RNA linkers 5’ + 3’

What is step 6 of CLIP-seq?

convert RNA to DNA

What is step 7 of CLIP-seq?

sequence DNAs

What is step 8 of CLIP-seq?

database searching/identification