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Advanced molecules, membranes and cells > Genomics and Proteomics > Flashcards

Genomics and Proteomics Flashcards

1
Q

Genomics

A

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

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2
Q

who coined the term genomics?

A

1986 - Thomas H Roderick

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3
Q

Functional genomics

A

DNA, RNA, proteins

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4
Q

largest known genome

A

amoeba

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5
Q

human genome project

A

start 1990 end 2003 still incomplete (centromeres)

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6
Q

what % genome encodes protein?

A

1.5

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7
Q

comparative genomics

A

evolutionary trees, carbon dating and molecular clock

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8
Q

what can be used to compare genomes?

A

USCS

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9
Q

what can be used to identify TFBS?

A

rVista

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10
Q

old method of finding where proteins bind to DNA

A

DNA footprinting

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11
Q

DNA footprinting

A

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

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12
Q

new way to find where proteins bind to DNA

A

ChIP sequencing

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13
Q

ChIP sequencing?

A

chromatin immunoprecipitation sequencing

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14
Q

2 requirements for ChIP

A

reference genome and reliable antibodies

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15
Q

ChIP - how is it done?

A

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

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16
Q

examples of epigenetic changes

A

DNA methylation, chromatin remodelling and gene silencing

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17
Q

when does methyltransferase work - epigenomics

A

add methyl when CG opposite methylated site

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18
Q

pharmacogenomics example

A

asthma - multifactorial, ARDB2 gene

19
Q

what can be used to identify pharmacogenomics?

A

DNA microarray - individual genes over or under expressed

GWAS - SNPs within genes that code for proteins

20
Q

proteomics - who coined term?

A

1994 Prof Marc Wilkins

21
Q

expression proteomics

A

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

22
Q

functional proteomics

A

identify protein-protein interactions, protein function

23
Q

post translational modifications

A

methylation, acetylation, ubiquination, phosphorylation

24
Q

what needs to be done to identify all proteins?

A

extract from cells/tissues using detergents and physical means

25
Q

detergents

A

break open lipid bilayers

26
Q

physical disruption

A

homogenise or sonicate

27
Q

homogenise

A

break cell walls and good for tissue samples

28
Q

sonicate

A

disrupt tissue wall and break open all cell membranes

29
Q

how are organelles isolated from cytoplasm?

A

centrifugation - density gradient

simple –> large organelles eg nuclei

30
Q

What does SDS do?

A

unfolds proteins and coats with NEGATIVE charge

31
Q

mobility in SDS-PAGE depends on what?

A

mass - small proteins go to the front

32
Q

what bonds does SDS break?

A

all covalent bonds - hydrophobic interactions

33
Q

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

A

coomassie brilliant blue or silver

silver 10x more sensitive

34
Q

what is used as the reference in SDS-PAGE?

A

standards of known mass to estimate mass due to migration

35
Q

what do 2D gels separate based on?

A

size and charge

36
Q

application of 2D gels

A

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

37
Q

what to do if protein gels but no reference?

A

HPLC
MALDI-TOF-MS
LC-MS/MS

38
Q

HPLC

A

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

39
Q

HPLC - mobile phase and how it works

A

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

40
Q

application of HPLC

A

newborn screening sickle cell anaemia - Hb

41
Q

MALDI-TOF-MS

A

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

42
Q

LC-MS/MS

A

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

43
Q

protein microarrays

A

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

Advanced molecules, membranes and cells (20 decks)

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