Genomics and Proteomics Flashcards
Genomics
study of all nucleotide sequences in chromosomes, including structural genes, regulatory sequences and non-coding DNA
who coined the term genomics?
1986 - Thomas H Roderick
Functional genomics
DNA, RNA, proteins
largest known genome
amoeba
human genome project
start 1990 end 2003 still incomplete (centromeres)
what % genome encodes protein?
1.5
comparative genomics
evolutionary trees, carbon dating and molecular clock
what can be used to compare genomes?
USCS
what can be used to identify TFBS?
rVista
old method of finding where proteins bind to DNA
DNA footprinting
DNA footprinting
ds DNA known sequence
label with radioactive P, add TF and stick to TFBS
cut DNA with restriction enzymes - cannot cut where protein is bound - control compared
new way to find where proteins bind to DNA
ChIP sequencing
ChIP sequencing?
chromatin immunoprecipitation sequencing
2 requirements for ChIP
reference genome and reliable antibodies
ChIP - how is it done?
DNA+proteins, treat with formaldehyde to link
sonicate to break open cells and smash DNA into fragments, add antibody specific for protein
magnetic beads bind antibodies and wash rest away
DNA purified using phenol/chloroform extraction
high throughput sequencing
examples of epigenetic changes
DNA methylation, chromatin remodelling and gene silencing
when does methyltransferase work - epigenomics
add methyl when CG opposite methylated site
pharmacogenomics example
asthma - multifactorial, ARDB2 gene
what can be used to identify pharmacogenomics?
DNA microarray - individual genes over or under expressed
GWAS - SNPs within genes that code for proteins
proteomics - who coined term?
1994 Prof Marc Wilkins
expression proteomics
levels of proteins, quantify proteome in 2 or more states eg healthy and disease
functional proteomics
identify protein-protein interactions, protein function
post translational modifications
methylation, acetylation, ubiquination, phosphorylation
what needs to be done to identify all proteins?
extract from cells/tissues using detergents and physical means
detergents
break open lipid bilayers
physical disruption
homogenise or sonicate
homogenise
break cell walls and good for tissue samples
sonicate
disrupt tissue wall and break open all cell membranes
how are organelles isolated from cytoplasm?
centrifugation - density gradient
simple –> large organelles eg nuclei
What does SDS do?
unfolds proteins and coats with NEGATIVE charge
mobility in SDS-PAGE depends on what?
mass - small proteins go to the front
what bonds does SDS break?
all covalent bonds - hydrophobic interactions
what stains are used to see the bands in SDS-PAGE?
coomassie brilliant blue or silver
silver 10x more sensitive
what is used as the reference in SDS-PAGE?
standards of known mass to estimate mass due to migration
what do 2D gels separate based on?
size and charge
application of 2D gels
change sin response to drugs in the proteome to determine side effects of a drug even when no cellular changes eg hepatomegaly in rats and pyrimidine
what to do if protein gels but no reference?
HPLC
MALDI-TOF-MS
LC-MS/MS
HPLC
separate proteins that are similar - can separate based on range of properties
HPLC - mobile phase and how it works
solvent - sample injected and detected - quantify monomers, tetramers and dimers
application of HPLC
newborn screening sickle cell anaemia - Hb
MALDI-TOF-MS
sample hit with laser to vaporise, charged ions accelerated by electrical gradients, large particles hit detector last
mass/charge vs intensity
LC-MS/MS
liquid chromatography separate proteins - select ion and break down further into fragments and another round of MS
protein microarrays
antibodies on array allow detection of many proteins with different properties in parallel
find protein complexes or binding partners
