Pharm Research: Gene Array and Proteonomics

Dr. Mageed
I. Describe and define various branches of functional genomics.
a. Genome (Genomics)
i. DNA
ii. Complete description of genes in an organism
b. Transcriptome (Transcriptomics)
i. RNA
ii. Complete description of mRNA in a genome.
c. Proteome (Proteomics)
i. Proteins
ii. Complete description of proteins expressed by a genome
d. Metabolome
i. Metabolites
ii. Complete description of metabolites expressed by a genome.
II. Give details on elements required to successfully construct a
gene array chip.
a. Ordered array small collections or spots of elements arranged in
uniformed rows and columns.
b. Microscopic elements
i. Collections of target molecules (DNA, cDNA, mRNA, proteins)
that allow specific binding of probe molecules (genes and gene
products).
ii. Must be smaller than 1mm
c. Planar surface parallel and unbending support
d. Specific binding single gene specificity to avoid cross-reactivity.
III. What are the advantages of using gene array over conventional
techniques?
a. Allows simultaneous monitoring of the expression level of genes of
gene products in parallel in different tissues, cells, and developmental
stages.
b. Generates LARGE and amounts of data with very little experimental
time, which is the opposite of traditional techniques.
IV. State the types of microarrays and explain their use.
a. Nucleic acid microarrays
i. Complementary (c)DNA microarrays
1. Commonly used (65% of publications)
2. Generates mRNA
3. Provide intense hybridization signals
ii. Oligonucleotide Microarrays
1. 26% of all publications
2. generates oligonucleotides
3. high specificity and good signal strength
b. Tissue and Protein Microarrays
i. 10% of publications
ii. contain sections from tumors or other tissues of interest
iii. contain pure protein or cell extracts
V. Describe in details all steps involved in performing a gene
array experiment (5).
a. A Biological Question and Array Fabrication.
i. Should be formulated b/f embarking on a microarray analysis
ii. Helps
1. Identify potential pitfalls
2. Assists in selecting controls and data analysis for array
fabrication.
b. Sample Preparation
i. DNA and RNA isolation and purification
ii. Target synthesis
iii. Probe amplification and preparation
c. Biochemical Reaction
i. Hybridization (DNA microarrays) and protein-protein
interactions (protein microarrays)
ii. Involves incubation of fluorescent sample w/ the microarray to
allow biochemical interactions b/w targets and probe
molecules.
d. Detection capturing and image from microarray using a scanning or
imaging instrument.
e. Data Analysis and Modeling quantitative and clustering analysis of
the data using imaging software.
VI. Discuss advantages/limitations of techniques involved in
probe preparation.
a. Coupling of an amplification strategy with reverse transcription
i. Method is efficient and reproducible
ii. Drawbacks:
1. Lack of linear amplification of all transcripts introduced
by too many PCR cycles causes the relative abundance
of cDNA products to be not well correlated with original
mRNA levels.
2. Most oligo (dT) labeling has significant 3-end bias.
b. Multiple rounds of cDNA amplification using in vitro transcription
i. T7 cycles can be repeated until enough cRNA is produced -
advantage
ii. Generates full-length products - advantage
VII. Describe the definitions of technology platforms (4) for
proteomics.
a. Bridges the gap b/w gene and product
b. Structural Proteonomics
i. Sample Preparation and Handling purification and isolation
ii. Peptide Mapping (obtaining protein information)
1. determination of partial amino acid sequence and
peptide mass.
2. MS, edman degradation.
c. Functional Proteonomics
i. Protein Identification and Quantificaton using information
1. Determining amino and carboxy terminal sequences
2. Peptide mapping and MS
ii. Cell Mapping
1. Protein-protein interactions
2. 3-D structure
3. Cellular localization
4. Post translational modifications
VIII. Give details on how to analyze proteins by a Mass
Spectrometer.
a. Components of MS
i. Ionization source excites solution or solid => gas phase ions
ii. Mass analyzer separates the gas phase ions according to
mass/charge ratio.
iii. Ion Detector measures the mass/charge ratio of each ion.
b. Can easily use proteolytically generated peptides recovered from gels
and membranes.
c. Peptide Mass Fingerprinting (PMS)
i. Uses trypsin the cleave proteins at Arg and Lys AAs
ii. Small # of peptides generated usually yield sufficient
information to permit protein identification in a protein
sequence database.
d. PMS is better for identification of proteins separated by 2D gels, while
information about protein M.W. and isoeletric points can be used to
aid in identification.
e. In contrast to peptides, mol. Mass of intact proteins are usually
insufficient to allow database identification.
IX. Describe steps involved in protein microarray analysis.
a. Protein Labeling - Proteins labeled with Cy3 or Cy5, followed by
removal of unincorporated dye via size exclusion chromatography,
dialysis, or affinity chromatography (if pt contains affinity tag)
b. Probing and Scanning The Array
i. Fluorescently labeled pt is dissolved in a buffer to minimize
non-specific binding
ii. Apply probing solution and incubate for 1 hr
iii. Wash slides successively and centrifuge to remove excess
buffer
iv. Images captured with a fluorescence scanner.
v. Spots of fluorescent intensities are indicative of the extent of
protein-capture molecule interactions.