Genomics and Proteomics

Question 1

Genomics

Answer 1

study of all nucleotide sequences in chromosomes, including structural genes, regulatory sequences and non-coding DNA

Question 2

who coined the term genomics?

Answer 2

1986 - Thomas H Roderick

Question 3

Functional genomics

Answer 3

DNA, RNA, proteins

Question 4

largest known genome

Answer 4

amoeba

Question 5

human genome project

Answer 5

start 1990 end 2003 still incomplete (centromeres)

Question 6

what % genome encodes protein?

Answer 6

1.5

Question 7

comparative genomics

Answer 7

evolutionary trees, carbon dating and molecular clock

Question 8

what can be used to compare genomes?

Answer 8

USCS

Question 9

what can be used to identify TFBS?

Answer 9

rVista

Question 10

old method of finding where proteins bind to DNA

Answer 10

DNA footprinting

Question 11

DNA footprinting

Answer 11

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

Question 12

new way to find where proteins bind to DNA

Answer 12

ChIP sequencing

Question 13

ChIP sequencing?

Answer 13

chromatin immunoprecipitation sequencing

Question 14

2 requirements for ChIP

Answer 14

reference genome and reliable antibodies

Question 15

ChIP - how is it done?

Answer 15

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

Question 16

examples of epigenetic changes

Answer 16

DNA methylation, chromatin remodelling and gene silencing

Question 17

when does methyltransferase work - epigenomics

Answer 17

add methyl when CG opposite methylated site

Question 18

pharmacogenomics example

Answer 18

asthma - multifactorial, ARDB2 gene

Question 19

what can be used to identify pharmacogenomics?

Answer 19

DNA microarray - individual genes over or under expressed

GWAS - SNPs within genes that code for proteins

Question 20

proteomics - who coined term?

Answer 20

1994 Prof Marc Wilkins

Question 21

expression proteomics

Answer 21

levels of proteins, quantify proteome in 2 or more states eg healthy and disease

Question 22

functional proteomics

Answer 22

identify protein-protein interactions, protein function

Question 23

post translational modifications

Answer 23

methylation, acetylation, ubiquination, phosphorylation

Question 24

what needs to be done to identify all proteins?

Answer 24

extract from cells/tissues using detergents and physical means

Question 25

detergents

Answer 25

break open lipid bilayers

Question 26

physical disruption

Answer 26

homogenise or sonicate

Question 27

homogenise

Answer 27

break cell walls and good for tissue samples

Question 28

sonicate

Answer 28

disrupt tissue wall and break open all cell membranes

Question 29

how are organelles isolated from cytoplasm?

Answer 29

centrifugation - density gradient | simple --> large organelles eg nuclei

Question 30

What does SDS do?

Answer 30

unfolds proteins and coats with NEGATIVE charge

Question 31

mobility in SDS-PAGE depends on what?

Answer 31

mass - small proteins go to the front

Question 32

what bonds does SDS break?

Answer 32

all covalent bonds - hydrophobic interactions

Question 33

what stains are used to see the bands in SDS-PAGE?

Answer 33

coomassie brilliant blue or silver | silver 10x more sensitive

Question 34

what is used as the reference in SDS-PAGE?

Answer 34

standards of known mass to estimate mass due to migration

Question 35

what do 2D gels separate based on?

Answer 35

size and charge

Question 36

application of 2D gels

Answer 36

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

Question 37

what to do if protein gels but no reference?

Answer 37

HPLC MALDI-TOF-MS LC-MS/MS

Question 38

HPLC

Answer 38

separate proteins that are similar - can separate based on range of properties

Question 39

HPLC - mobile phase and how it works

Answer 39

solvent - sample injected and detected - quantify monomers, tetramers and dimers

Question 40

application of HPLC

Answer 40

newborn screening sickle cell anaemia - Hb

Question 41

MALDI-TOF-MS

Answer 41

sample hit with laser to vaporise, charged ions accelerated by electrical gradients, large particles hit detector last mass/charge vs intensity

Question 42

LC-MS/MS

Answer 42

liquid chromatography separate proteins - select ion and break down further into fragments and another round of MS

Question 43

protein microarrays

Answer 43

antibodies on array allow detection of many proteins with different properties in parallel find protein complexes or binding partners