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Flashcards in Genome Wide Regulation Deck (25):
1

GeneArrays

used to measure the amount of mRNA from all the genes in an organism

2

RNA-seq

Uses next generation DNA sequencing to determine transcriptome

3

2-D gels

used to measure the amount of proteins expressed

4

Mass-spectroscopy

identifies proteins

5

Gene Arrays

- Each gene of a sequenced organism is fixed to a slide in an ordered "array"
- the army is then probed with mRNA from your cell (two conditions)

6

Gene Array Technique

- RNA Isolation
- cDNA Generation
- Labeling of Probe
- Hybridization to Array
- Imaging

7

Campylobacter has how many genes

1600

8

Making the array

- genes or portions of genes are spotted in duplicate on glass slides by robots.

9

Probing the array

- microarrays are good at comparing transcription profiles between two different conditions
- First, you make cDNA copies of the RNA with reverse transcriptase
- cDNA from one condition is labeled red dye
- The other condition is labeled with green dye
- Both sets are then hybridized to the array, and a machine detects the color.

10

Gene Arrays

- then probed the away
- each gene on there twice
- yellow-both genes expressed

11

Heat Map

- shows how much red versus gene per green
- repeat experiment and swap dyes looking for exact opposite effect
- fold changes never really greater than 3-4
- only so green.

12

RNA seq

- total RNA
- eukaryote (enrich mRNA by oligo(dT))
- prokaryote (remove RNA by kit)
- RNA fragmentation
- Random hexameter primed cDNA synthesis
- Ends repairment, adapter ligation, and PCR amplification
- Hiseq 2000 91PE or 101PE sequencing

13

Flagella discussion

- RpoN mutant
- RNA isolated from both conditions
- DNA digested by DNase
- rRNA depleted
- cDNA made with random primers
- RpoN required for Flagella (class II genes)
- cjc242 is a host invasion space gene that uses hook apparatus to invade host cells

14

Proteomics

the study of all the proteins in a cell

15

advantages of proteomics

detects non-transcriptional control, can be cheaper

16

disadvantage of proteomics

cheap methods miss low abundance proteins

17

1st dimension of 2D electrophoresis

- separates by charge (isoelectric focusing)
- the charge on a protein depends on PH
- isolelectric point is pH where they have no charge
- proteins with positive charge will migrate toward negative electrode until they lose enough protons to lose charge

18

2nd dimension of 2D electrophoresis

- separates by size (SDS PAGE)
- each spot corresponds to size

19

Ahpc

highly expressed protein

20

NapA

compensates for loss of Ahpc

21

determining the spots

- spots that are regulated are cut out of the gel and extracted
- to determine protein identity:
- Do N-terminal sequencing
- determine the mass of protein by mass spectrometry.

22

Tryptic Digest

- trypsin cuts proteins after argentine and lysine residues
- excise the spot, treat the spot with trypsin to cut it up
- weight fragments by mass spectroscopy

23

LC-Maldi TOF

- Tags differ by 1 amu
- mass to charge ration (M/Z) is determined by the amount of time it takes to get to the detector - Time of Flight
- keep the charge 1, you get the mass
- time to get from pusher to detector tells you how big
- fly for about 12 feet

24

Mass spectrum

every fragment that comes through we weigh

25

computer puts together the sizes

the computer program determines the sizes of all the proteolytic fragments from the predicted genome sequence
- computer compares experimental mass spectrum to the predicted mass spectrum from the database
- identifies the protein that the fragment came from.