9/17/21

Lecture 8: Bioinformatics and Omics

1. Northern blotting
a. Antiquated
b. Detects RNA
i. Gel electrophoresis
1. Electric current runs through, charges the RNA molecules,
separates
ii. Transfer RNA from gel to nylon membrane
iii. Nylon membrane prepared with bound RNA fragments
iv. Incubate membrane
1. Labeled probe to allow hybridization
v. View autoradiogram
1. Shows location of RNA molecules
2. Bioinformatics
a. Generating large datasets to answer multiple questions simultaneously
i. Rapidly expanding field
b. Genomics
i. Study of genome
ii. Human Genome Project
1. Sequenced 3.3 billion base pairs of the human genome
3. Omics
a. Scientific approach
b. Considers all members of a certain class of molecules/interactions collectively
c. Terminology
i. Transcriptome
1. All RNA molecules in an organism, cell, or tissue
2. Varies across life span/location
ii. Proteome
1. Collection of all proteins in organisms, cell, or tissue
iii. Kinome
1. All phosphorylation modifications in cell
iv. Lipidome
1. All hydrophobic molecules in cell
v. Glycome
1. All carbs
vi. Metabolome
1. All small metabolites in organisms, cell, or tissue
vii. Interactome
1. All interactions of molecules in cell
4. Techniques
a. PCR (Polymerase Chain Reaction)
i. Denature DNA by heating to 95°C
 ii. Lower temp to 50-60°C
1. Allow primers to anneal to complementary DNA strands
iii. Raise temp to 72°C
iv. Start over
b. Fluorescent sequencing
i. Fluorescent dideoxy terminators
1. Dideoxy has 2 -OH’s
a. No new nucleotides can be added to it (aka, terminator)
ii. Reads 700+ bases per reaction
c. High-throughput parallel sequencing
i. Sequence+assemble entire genomes within days
ii. Short DNA chunks are sequenced and then assembled into whole
genome using existing genomes as scaffolding
d. Microarrays
i. Probing transcriptome
ii. cDNA’s corresponding to specific gene are robotically spotted onto a
microscope slide
iii. RNAs purified from experimental/control samples
1. cDNA copy made from RNAs using reverse transcriptase
iv. Each RNA sample labeled with dye
e. Serial Analysis of Gene Expression (SAGE)
i. Don’t need to know sequence ahead of time
ii. Data is more exact than microarrays
iii. Expensive, time consuming, and produces less data
f. RNA sequencing (RNA-Seq)
i. AKA whole transcriptome shotgun sequencing (WTSS)
ii. Analysis of transcriptome!!
1. Provides info about which genes are being actively transcribed at
a particular moment and the relative abundance of the
transcript
iii. Initial steps:
1. Isolate mRNA
2. Transcribe to cDNA
3. Strands amplified & adapters ligated to ends
4. Sequence & analyze strands
5. Mass spectrometry
a. Used to quantify proteins, lipids, carbs, and metabolites
b. Steps
i. Volatile sample ionized
1. Soft techniques
a. Electrospray ionization (ESI)
b. Matrix-assisted laser desorption ionization (MALDI)
ii. Charged molecules separated based on mass-to-charge ratios
1. Types of mass analyzers:
 a. Quadrupole
b. Time-of-flight mass analyzer
c. Fourier transform mass spectrometer
iii. Molecules resolved in EM field and travel to detector
1. Electron multiplier or microchannel plate detector
c. Other mass spec techniques
i. Liquid chroma
ii. SDS-PAGE or 2D PAGE
iii. Peptide mass fingerprinting
iv. Tandem mass spec (MS-MS)
v. Collision-induced dissociation (CID)
6. GenBank
7. Protein Data Bank (PDB)
8. Sequence alignments
9. Search algorithm
10. BLAST
a. Altschul algorithm
b. Must know: how all BLAST types work
11. Interactome
a. Map showing which proteins interact with each other
b. Not good for auto-generation