BIOL-UA 21 Lecture Notes - Lecture 9: Agarose Gel Electrophoresis, Northern Blot, Southern Blot

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copperclam97

18 Mar 2016

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Recombinant dna iii: molecular analysis using cloned sequences. Detecting a dna sequence in a complex mixture: Extract dna which is cleaved by restriction enzymes and run on agarose gel. Dna fragments are blotted onto nitrocellulose filter. Filter is exposed to radioactive probe and wash whatever it can base pair with will stay and whatever doesn"t goes away. Cut with ecori or xbal and then perform a southern blot and tell the difference between the normal sequence and the mutant sequence. Detecting an rna sequence in a complex mixture. Extract mrna and run on agarose gel. Filter is exposed to radioactive probe either dna or rna probe and will wash whatever it can base pair with will stay and whatever doesn"t goes. Filter is exposed to photographic film; autoradiogram. Northern blot detects changes in gene activity: Leukemia cells can be induced to stop growing and differentiate into erythroblasts (precursor of mature red blood cells) After induction of beta-globin synthesis increases 1000-fold.

Related Questions

1. A study was conduced to identify a liver cell gene (zed) that has a length of 3000 bp. A southern blot was prepared using DNA from the liver cell and a probe made to region 1600-2000 of zed. Analysis of the blot revealed that there are two spots present, one at 3000 and another at 2000. A plausible explanation for this observation is

A. there is an identical copy of this gene on another chromosome.

B. the gene was cut into two equal portions by a restriction enzyme.

C. there is another gene with sequence similar to gene zed

D. both: there is an identical copy of this gene on another chromosome and there is another gene with sequence similar to gene zed

E. no explanation can be given using the available data

2. Protein P is synthesized in relatively high amounts in human pancreas. This protein has been isolated and purified, but its amino acid sequence has not been determined. We wish to clone the gene for protein P.

(a) How can a probe be prepared to identify the gene for protein P?

(b) If we have prepared a radioactive messenger RNA as our probe in part(a), how could we verify that it is the mRNA for protein P?

(c) If we wish to make a gene library from the DNA of human pancreatic cells, we proceed by digesting the DNA with the restriction enzyme BamHI and then separating the fragments by agarose gel electrophoresis. The fragments are transfered (by southern blotting) onto a nitrocellulose filter and flooded with the radioactive probe. Following incubation, the filter is washed to remove any probe that has not specifically bound to one or more DNA fragments. Autoradiography of the filter reveals two bands of size 300 and 700 bp. When experiment is repeated using restraction enzyme PstI, only one band of 1000 bp appears. If it is already known that pancreatic cells contain a single copy of the gene of protein P, which of these enzymes should be used to construct our gene library?

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BIOL-UA 21 Lecture Notes - Lecture 9: Agarose Gel Electrophoresis, Northern Blot, Southern Blot

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