Differential gene expression

Question 1

Southern blot analysis 5 steps

Answer 1

1. Digest genomic DNA with specific enzymes into fragments 2. Separate fragments by size using gel electrophoresis 3. Transfer fragments to membrane perpendicular to original direction of separation to allow fragments to bind membrane surface and easily accessed 4. Transferred DNA probed (hybridized) with labeled probe 5. Signal from label identifies DNA fragment

Question 2

Northern blot analysis (2 differences from Southern blot)

Answer 2

Identical to Southern blot except: 1. RNA is used as test material 2. RNA sample not digested by enzymes

Question 3

5 main advantages of Northern blot

Answer 3

1. Proportional annealing of probes (if know amount of label know amount of mRNA) 2. High specificity (hybridization for single mRNA should yield one band on membrane 3. Quantitative (measure against control housekeeping genes) 4. Reliable 5. Membranes with probes reusable

Question 4

3 disadvantages of Northern blot

Answer 4

1. Long process 2. Low sensitivity (needs large numbers of mRNA) 3. Low yield (one gene per hybridization)

Question 5

Polymerase chain reaction (PCR)

Answer 5

Amplifies DNA exponentially using mRNA as source material-converted to cDNA used in PCR. Theoretical yield=(2^n)x where n=# cycles, x=original copy # Extra step to generate cDNA using reverse transcriptase needed.

Question 6

3 types of primers in PCR

Answer 6

1. Random (will bind anywhere and then reverse transcribes into cDNA) 2. Oglio (only binding on one end of mRNA, transcribes along full length of strand) 3. Sequence-specific (binds only one particular sequence)

Question 7

PCR template

Answer 7

DNA sample to be amplified

Question 8

Primers

Answer 8

Small synthetic single stranded DNA molecules that are complimentary to either end of region to be amplified

Question 9

Thermocycler

Answer 9

Heating/cooling device

Question 10

4 steps of PCR

Answer 10

1. Denaturing step (mixture heated to separate strands) 2. Annealing step (Cooled to temp that primers will bind to separated strands) 3. Extension step (temp brought to room temp where DNA polymerase efficient, primers extended and doubles numbers of double stranded DNA) 4. Steps 1-3 repeated

Question 11

Real time polymerase chain reaction (quantitative)

Answer 11

Uses fluorescent dyes to monitor progress of PCR reaction

Question 12

Real time polymerase chain reaction (quantitative) 4 steps

Answer 12

1. Set up conventional PCR protocol 2. Add syber green dye to reaction mix 3. Perform amplification on fluorescent thermal cycler 4. Monitor reaction and process data to yield quantity

Question 13

In situ hybridization

Answer 13

Localization of DNA sequences or gene products (mRNA) in order to study spacial characteristics within tissue (find out where gene being expressed)

Question 14

Positive control

Answer 14

Phenomenon is expected–ensure an effect where there should be an effect

Question 15

Negative control

Answer 15

No phenomenon expected–ensure no effect where there should be no effect.

Question 16

5 requirements for in situ hybridization

Answer 16

1. Preservation of mRNA 2. Tissue morphology 3. Tissue sectioning 4. Hybridization (isotopic and non-isotopic) 5. Detection

Question 17

Microarray analysis

Answer 17

Allows for simultaneous evaluation of multiple genes. Similar to Northern blot but in reverse (solid support is now the probe). Oglionucleotides or cDNAs for known expressed genes placed on membranes, slides, silicone chips hybridized to labeled probes. Suited for comparing 2 or more different populations of cells or tissues.

Question 18

4 microarray steps

Answer 18

1. Isolate RNA from 2 samples 2. Labeled cDNA probe generated 3. cDNA for gene on solid support 4. Detection by laser scanning

Question 19

3 microarray outcomes

Answer 19

1. Yellow: no differential signal (shared by both samples) 2. Red: overexpression in treated signal 3. Green: underexpression in treated signal

Question 20

4 disadvantages to microarray

Answer 20

1. Expensive 2. Reliability (still in development) 3. Data overload (huge amount of information) 4. Only useful for known genes and products

Question 21

Differential display

Answer 21

Compare 2 different RNA populations. PCR based technique using random primers for amplification of cDNAs in different populations

Question 22

Subtractive hybridization

Answer 22

PCR based technique using subtractive and selective amplification to view genes unique to tester population

Question 23

3 steps of differential display

Answer 23

1. RT-PCR performed on 2 populations of RNA using same set of primers with no real specificity (RNA–>cDNA–>Amplified DNA products) 2. Products run out on separating gel and compared 3. Different bands isolated from gel and sequenced and characterized

Question 24

Advantage of differential display

Answer 24

Large number of differentially expressed genes can be seen on a single gel

Question 25

Disadvantage of differential display

Answer 25

Large numbers of primers needed to see ALL differently expressed genes in population

Question 26

Cluster analysis

Answer 26

Looking for whether a family of genes changes over course of time or conditions