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DesLee26
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The human beta-globin genes provide an excellent example of __
A differentially expressed gene is switched on only when __
At all other times the gene is __
- differential gene expression
- the biological information that it contains is needed
- inactive
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With beta-globin genes, the differential expression profile is linked to __, but other genes display __, being active in some cell types but inactive in others
- development
- tissue specificity
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Knowing in which tissues a newly discovered gene is active often provides the first clue that leads eventually to __
an understanding of the function of that gene
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A gene’s mRNA can be detected by __
Tissue extracts can be tested for the presence of a particular mRNA by __
hybridization probing x2
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The basis of this technique is the ability of __. This can occur not only between __, but also between __
The formation of base pair between any two polynucleotides is called __
- any two complementary polynucleotides to form base pairs with one another
- ssDNA to form the double helix
- combinations of one DNA and one RNA strand
- nucleic acid hybridization
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ii. Steps of hybridization:
1 and 2
Prepare the sample of RNA from the tissues being studied Spot onto a nylon membraneà RNA in sample becomes bound to membrane
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ii. Steps of hybridization:
Steps 3 and 4
To determine which samples contain a particular mRNA, the membrane is probed with DNA whose sequence matches that of the gene we are interested in
It can be a segment of DNA or an oligonucleotide with the same sequence
Hybridization occurs
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The probe must be labeled so it can be detected after hybridization.
What is one way this can occcur; and, what is a problem with it?
One way: radioactive phosphorus, which is incorporated into the sugar-phosphate backbone of a DNA molecule, and then analyze via autoradiography, in which a sheet of X-ray-sensitive film is placed over the membrane. The samples that contain hybridized RNA are dark spots Problem: hazardous when disposed
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The probe must be labeled so it can be detected after hybridization.
What is a second way?
Alkaline phosphatase attached to probe DNA Detection by degradation of phosphatase with luminol, which results in emission of light, which can be recorded on normal photographic film
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Expression profiling was used to ID the CFTR gene
Initially, they identified a candidate gene, one they thought might be the one they were looking for. __ was a test to confirm if it was the CFTR gene
Expression profiling
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What was done to profile the CFTR gene?
- 1) RNA extracts prepped from various tissues
- 2) They were probed with a clone of the candidate gene (which was radioactively labeled)
- 3) They were developed to show that the probe hybridized with RNA extracts from teh pancreas and nasal polyps
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