Biology 3338A Lecture Notes - Lecture 4: Northern Blot, Southern Blot, Syncytium

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limehorse569

22 Aug 2016

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To ensure selected mice are homozygous recessive, do southern blot. Isolate genomic dna and put through gel smaller dna travels further down while bigger dna does not go far from the top. Bmp probe is radioactive-bmp dna that is complementary to bmp dna in genomic dna on gel. Wt bmp probe creates one band on gel signifies both alleles are of same size and smaller since further down. Heterozygous bmp probe creates two bands on gel signifies different alleles of which smaller band matches wt band and bigger band matches bmp7- band. Mutant band is bigger because added in neor gene. Homozygous recessive bmp probe creates one band on gel signifies both alleles are of same bigger size. Another probe that can be used is radioactive-neor probe ie. neor dna that is complementary to neor dna in genomic dna on gel. Only heterozygous and homozygous recessive have bands as only they have bmp7- allele with the neor gene.

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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Biology 3338A Lecture Notes - Lecture 4: Northern Blot, Southern Blot, Syncytium

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