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Flashcards in Differential gene expression Deck (26):
1

Southern blot analysis 5 steps

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

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Northern blot analysis (2 differences from Southern blot)

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

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5 main advantages of Northern blot

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

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3 disadvantages of Northern blot

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

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Polymerase chain reaction (PCR)

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.

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3 types of primers in PCR

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)

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PCR template

DNA sample to be amplified

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Primers

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

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Thermocycler

Heating/cooling device

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4 steps of PCR

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

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Real time polymerase chain reaction (quantitative)

Uses fluorescent dyes to monitor progress of PCR reaction

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Real time polymerase chain reaction (quantitative) 4 steps

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

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In situ hybridization

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

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Positive control

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

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Negative control

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

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5 requirements for in situ hybridization

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

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Microarray analysis

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.

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4 microarray steps

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

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3 microarray outcomes

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

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4 disadvantages to microarray

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

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Differential display

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

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Subtractive hybridization

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

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3 steps of differential display

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

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Advantage of differential display

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

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Disadvantage of differential display

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

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Cluster analysis

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