Genomics, Proteomics, Transcriptomics M.Bio Block 3
What are the separation techniques of proteins?
1D Gel Electrophoresis 2D Gel Electrophoresis Protein Digest Purification Isotope Coded Affinity Tags (ICAT) 5 ways
Describe process of 2D Protein Electrophoresis. Define Isoelectric Point.
2 Dimensional Gel Electrophoresis: First separation = Charge (Isoelectric Point) Second Separation = Mass Isoelectric point = When Protein has Zero Charge (neutral)
What are the methods to detect a SINGLE GENE TRANSCRIPTIONAL change?
3 methods: Hybridization Based: Northern Blotting: Identifies TARGET mRNA species (Transcript specific). PCR Based: Exponential amplification of initial differences in transcript number. Reporter gene-based: Fusions of a promoter of a gene of interest with reporter gene (GUS), green fluorescent protein or luciferase. Activity measured via fluorescence.
Describe DNA microarray. Identify different types of microarrays
4 Different types: Comparative Genome Hybridization: Compare pt DNA to Control DNA. Looking for Missing/duplicated regions. Pt DNA + Control DNA COMPETE to attach pre bound complimentary NT seq in Microarray. Results: Equal DNA Loss DNA - more control binds vs pt. Gain DNA - more pt binds vs control. SNP Chips: Look for unique single base pair changes that are KNOWN. Use OligoNT Hybridization Analysis. Will not hybridize with target DNA that has a single base pair change. Very specific. (Sickle Cell dot blot) Positive signal indicates PRESENCE of particular SNP. Expression Arrays: Expression of mRNA in different tissue Tiling Arrays: Expression of mRNA in different tissues.
What are ASO's and SNPs
Allele Specific Oligonucleotide Probes - DNA marker resultin from a BASE PAIR difference at one particular site in genome. Use this for Sickle Cell. SNP = single base pair difference or change. ASO is a technique for looking at one SNP at a time.
Describe process of western blotting + specific protein identification + what probe it uses
Before Western Blotting: Need to separate protein via 1D or 2D Gel Electrophoresis. 1. Use SDS-PAGE to heat protein and make it negatively charged so that it will flow down a polyacrylamide gel. Also want to heat to REDUCE the suldfide bond between subunits of proteins. Proteins are STAINED WITH COMASSIE BLUE. DNA stained with Ethydium Bromide! 2. Transfer proteins to SOLID SUPPORT (nitrocellulose membrane) via ELECTROBLOTTER. Western Blot = resulting membrane containing transferred protein. Western Blot = allows you to identify SINGLE specific Protein. 3. Blocking = with 5% skim milk. 4. Incubate with Primary antibody, then Secondary Antibody + Horse Radish Protein which will allow chemiluminescence. Note: You can have many proteins that have the SAME SIZE/WEIGHT -> which is why you want to separate by IsoElectric Point in 2D Gel Electrophoresis.
Explain how CGH, SNP + NGS work (all under microarrays)
CGH: Comparative Genome Hybridization: - Copy number variations (each person has 2 copies of every AUTOSOME gene) - Compare pt DNA to Control DNA - look for abnormalities. Labeling through fluorescent dyes + applied to microarray and will hybridize. (DNA LOSS/GAIN/EQUAL HYBRIDIZATION) SNP Chips: DNA marker resulting from a BASE PAIR DIFFERENCE at one site in a GENOME. Use OligoNT hybridization analysis. Short OligoNT synthesized complimentary to common allele + hybridized under high temp to DNA samples. Positive signal = PRESENCE of SNP NGS: Next Generation Sequencing: Carrying out parallel sequencing. Millions of DNA fragments sequenced in UNISON. - Rapid sequencing whole genome + Low cost. - In Situ PCR + extending molecule. Benefits: Whole genome sequencing: Of different species + to identify genes involved in disease. Whole Exome Sequencing: Exome = ONLY protein coding regions. For discovering disease. Targeted Sequencing: Specifically targeting regions of genome of interest. MOST affordable + higher yield. Can provide better treatment options for patients + identify/differentiate new disease or disease subtypes.
Define Western Blot
Can be used to detect and quantify the amount of a PARTICULAR PROTEIN SNW DRP Southern = DNA Northern = RNA Western = Protein
What do typical proteomic experiments look for?
Comparing Spots for evidence of differnetial expression. Take cells grown in vitro and expose them to 2 different conditions. See which protein expressions differ between two conditions via 2DGE.
What are automated DNA Seq?
Computerized tests involving loading all 4 rxn products into a single lane and capturing seq. data. Allow for Faster sequencing.
What is Cytogenetics?
Diagnostics of chromosomal abnormalities. Karyotype Analysis detects: Euploidy: Normal Diploid number 46 chromosomes, 22 autosome pairs + 2 sex chromosomes. Aneuploidy: Number of chromosomes differ from normal. Only chromosomes compatible with life are: 1- Extra Chromosome 13,18,21 (patou, edwards, downs syndrome respec.) 2- Extra X/Y chromosome (Klinefelters syndrome) 3- Single X chromosome (Turner)
Define RFLP + FISH
FISH = Detect Microdeletions Example of metaphase cell hybridized with probe for Steroid Sulfatase Deficiency. It binds CENTROMERE on chromosome which is X Cen. Once it finds both X chromosomes, only the one that has Steroid Sulfatase gene signal will fluoresce. The chromosome with the deficiency will NOT fluoresce. RFLP = Restriction Fragment Length Polymorphism. 1 - isolate DNA and digest with restriction DNA 2 - resulting DNA separated via gel electrophoresis 3 - DNA fragments denatured + transferred to membrane 4 - membrane hybridized with labelled DNA fragment corresponding ot chromosomal region containing polymorphism Probe that is specific to DNA seq will bind and will create bands.
What is relationship between: Genome Trasncriptome Proteome as it relates to time and which cells are used as a study subject
Genome tells everything about the organism's life, development, disease resistance, metabolism to drugs, history + environment.
Define: Genome Transcriptome Proteome Genomics Gene DNA Seq SNP Allele Haplotype
Genome: Entire genetic compliment of an organism encoded in nucleic acids Transcriptome: Analysis of mRNA present in a cell at a particular time Proteome: Dealing with proteins Genomics: Study of genomes Gene: Unit of hereditary info made up of discrete DNA seq encoding proteins DNA Seq'ing: Process of determining the precise order of NTs within a DNA molecule Single Nucleotide Polymorphism: Polymorphism where the alleles vary within one single NT base. Allele: One of a number of alternative forms of a gene on same genetic locus Haplotype: Combination of aleles at adjacent locations on a chromosome that's inherited together.
What are some challenges of Proteomics in the genome and in Proteome?
Genome: Static with amplification possible, has homogeneous molecules with no variability. Proteome: Is dynamic, with NO amplification, having HETEROgenous molecules + large variability.
Describe shotgun approach
Genomic DNA cut into pieces of 150mb and inserted into BAC VECTORS, transformed into E.Coli where they are replicated + stored. Cleaving occurs twice. Shotgun seq is overlapping sequence based on cutting with different restriction enzymes.
What is Transcriptomics? Compare to Genomics?
Global analysis of gene expression (genome wide expression profiling). Genomics + Transcritomics address same biomolecules: Nucleic Acids + Simple informative structure + similar chemical properties. Transcriptomics uses REVERSE TRANSCRIPTION to study RNA. Uses Electrophoresis, Hybridization, sequencing.
What are the stages of Mass spec?
Introduce sample to Mass Spec. Generate ions in Gas phase using MALDI or ESI Separate ions via differences in mass to charge ratio. Detect ions + identify proteins via database.
What are Isotope Coded Affinity Tags? (ICAT) How do you acquire Protein Structure Info?
It labels protein samples from two different sources with two identical reagents that differ ONLY IN MASS. Does NOT DEPEND on separation of proteins by 2DE. Protein Structure information: Edman Sequencing - obtaining N-Terminal AA seq. Fragmented at Methionine OR Tryptophan (3-5 peptide fragments) Mass Spec: Sample prep/ionization/analysis
What is Proteomics?
Large scale characterization of ENTIRE PROTEIN complement of cell line/tissue or organism AT A GIVEN TIME! It is Time/Context dependent! Use mRNA analysis, genomics + yeast two-hybrid analysis.
What's the purification technique of peptides?
Liquid chromatography Capillary electrophoresis Cation exchange Reverse phase chromatography.
How do you digest proteins?
Mass Spectrometers + proteases cleave at specific AA. Example: Trypsin, Chymotrypsin
Describe Sanger/Dideoxy DNA seq rxn
Method to obtain the base pair sequence of a piece of DNA. Principle: 1- Based on extending DNA strands from a PRIMER bound to ssDNA template. 2- Rxn contains BOTH DEOXY-NTs + Labelled DIDEOXY-NTs + DNAP, primer, buffer. 3- Random incorporation of DIDEOXY-NT will terminate synth of DNA strand as ddNTP LACKS 3' FREE OH. ddNTP = NO free 3'OH. Sanger seq: 4 reactions conducted each containing different ddNTP. Based on termination of DNA chain extension by ddNTP. Sequence rxn: Primer binds comp. site on template strand. DNAP synth new strand by extending primer. Compl. NTs added. Incorporation of dNTP results in continued synth. Incorporation of labelled ddNTP results in termination + labelled DNA fragment. Banding pattern shows you where DNA terminated and what base it terminated at.
What does proteomics give?
One gene - multiple products via alternative splicing Genome annotation Protein expression/function studies Protein-Protein interactions
What are methods to detect MULTIPLE Gene Transcriptional Changes
PCR Based: Reverse Transcriptase. Divide cDNA pool into subsets + separate on gel, quantify band intensity. Hybridization Methods: Macroarray + Microarray. Small membrane (macroarray) or glass slide (microarray). Fluorescently label mRNA samples of interest - SINGLY OR COMPETITIVELY HYBRIDIZE TO A SLIDE. In Single experiment, expression levels of genes determined by measuring amount of mRNA bound to each gene on the array (COMPETITION)! Sequence Based: SAGE (Serial Analysis of Gene Expression) OR MPSS (Massively Parallel Signature Sequencing): - Short seq signatures made from defined position within mRNA. - relative abundance of these signatures represent quantitative estimate of expression of that gene - NO SEQ KNOWLEDGE REQUIRED! UNIVERSAL to study ANY transcript.
What are the ways to validate protein function?
Protein Function Microarrays: Screen various types of protein interactions: Protein - Protein, Lipid, DNA, drug + peptide interactions. Also used to identify enzyme substrates AND immune responses Phage Display Approach: Used to identify INTERACTING PROTEINS (Protein-Protein Interaction). Pair a fused protein with phage coat protein, if expression occurs the known protein is displayed IN PHAGE COAT. The interaction means that the UNKNOWN protein is LIKELY INVOLVED in cell-cell signalling. Yeast Two-Hybrid System: Hybrid 1 = BAIT protein (yeast domain with KNOWN human domain) Hybrid 2 = Activation domain (Yeast protein + unknown proteins, many of these) Screening for PROTEIN PROTEIN interactions If Hybrid 1 + Hybrid 2 bind together and RNA Polymerase is activated = Gene expression occurs therefore some factor in Hybrid 2 allows for gene expression to take place when it interacts with Hybrid 1. If no gene expression = Hybrid 2 does not have any qualities to help regulate gene expression when interacting with Hybrid 1.
What is the method of SAGE?
SAGE: Generates sequences from cDNA fragments for discovery of NEW GENES + quantification of their expression levels in a certain tissue. Method to detect MULTIPLE changes. Small pieces of DNA linked together to form a CONCATAMER (long chain). These seq Tags are 9-14bp in length, library may contain 50K tags.
When should you use each DNA Microarray method?
SNP Chips: Point mutations Microarrays: Gene expression Tiling Arrays: Expression across a genome or chromosome. Made up of HIGH DENSITY PROBES. Use array-based hybridization to scan. Identifying novel transcripts or exons. Able to identify regions of chromosomes that are TRANSCRIPTIONALLY ACTIVE.
What are Two Channel MicroArrays?
Similar microarray tech used to identify potential gene targets for therapies to treat human disease. Isolation of two tissue RNAs -> production of labelled cDNA -> Hybridization of labelled cDNA -> visualization of hybridization.
What is flourescence in situ hybridization?
This technique allows you to LOCALIZE large DNA fragments at METAPHASE OR INTERPHASE. DNA fragment labelled and hybridized to metaphase choromosomes. Label DNAprobes with particualr dye so that when you have chromosomes and it hybridizes with probes it will compl. bind all specific chromosomes probe made for. If 3 copies of chromosome 18, it will show 3 Chromosome 18s. Can also diagnose LARGE chromosomal mutations (duplications, deletions, rearrangements) - Standard karyotype (CANNOT resolve microdeletion) - Aneuploid Screen test (apply probe directly to cells which will bind specific chromosomes and flouresce) STAIN WITH GIEMSA TO SEE BANDS! Banding patterns are different for every chromosome.
What is the value of Transcriptomics?
Used to identify mRNAs + relative abundance. Gives an understanding of genes + pathways. Similar gene expression = under same control mech (guilt by ass). Helps solve function of unknown genes + identifies marker genes for diseases + expression allows indirect inferences about genetic differences + proxy for changes in proteome.
What are Expression Arrays?
cDNA array containing features representing a set of KNOWN expressed sequences. Used to identify a set of genes or ALL genes that are expressed in a cell. Used to COMPARE GENE EXPRESSION (transcription) between cell/tissue samples.
What is the method of MPSS?
cDNA fragments cloned into MICROBEADS using Lynx Megaclone Tech. Starting with 1million mRNA molecules -> Megaclone produces 1 mil beads, each containing 100K cloned copies of cDNA from each mRNA molecule. Molecules attached at polyA ends covalently so DPN2 end available. Possible without organism sequence info + counts the # of individual mRNA molecules representing each gene.
What are Microarrays?
cDNA microarray is based on the ability of a given mRNA to bind specifically to its original DNA coding seq in form of a cDNA template on the array. The cDNA seq has an EST (expressed sequence tag) that is used to identify gene transcripts. Affymetrix Gene chip = tech that allows this - causes fluorescent tags. Can be competitive in two color arrays or single in one color array.