01:447:384 Study Guide - Final Guide: Southern Blot, Northern Blot, Western Blot

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coffeegoat901

23 Apr 2020

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Complete the diagram to describe a southern blot procedural design. The southern blot is a technique used to detect a specific dna sequence in samples. One takes fragments of dna separated during gel electrophoresis to a filter membrane made of nitrocellulose or nylon filter by blotting. The filter is then exposed to radioactively labeled probe for a specific gene, then the probe hybridizes with a short sequence present on the gene. The filter is then exposed to x-ray film, then the fragment containing the gene of interest is identified by a band on the film. Gene arrangements can be analyzed, and specific fragments of dna can be identified within a mix of many other fragments. Also, the southern blot procedure can be used to identify specific dna fragments that might cause genetic disease, and be able to study it. There are two related procedures that use the same principle of the southern blot.

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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?

violethornet799

Please answer the following multiple-part question regarding probe synthesis and nucleic acid hybridization:

a) You would like to determine how many genes are expressed in the normal hepatocytes (liver parenchymal cells) of a healthy person and the hepatocytes of an alcoholic. Which of the following blots would you utilize and how many hybridization experiments you must perform to complete the experiment?

A. A Sothern blot (membrane-based)/two

B. A Northern blot (membrane-based)/one

C. A microarray/two

D. A gene chip/one

b) Of the two genes, one has a sequence of 5âCCCAUUUGG3â and another has the sequence 5âCCCAAUUGG3â. You want to distinguish the two genes by Southern blot hybridization. Which of the following parameters you may not have to control?

A. The membrane to be used (nylon membrane versus PVDF membrane)

B. Detergent (i.e. SDS) concentration in the hybridization buffer

C. Presence of non-specific DNA in the hybridization buffer

D. Temperature of the hybridization buffer

c) Solid-phase hybridization is different from solution hybridization because of all these, except______.

A. the former uses probes, the later do not

B. the later may use enzymes, the former donât

C. the former uses membrane, the later donât

D. the former immobilize nucleic acids, the later donât

Huntington's disease is an inherited neurological ailment with a variable age of onset. A protein called huntingtin has a sequence of repeated glutamine residues, all encoded by CAG. The number of repeated CAG triplets is expanded in Huntington's disease, apparently as the result of replication errors. The age of onset is related to the number of CAG triplets in the repeat region; the more glutamine codons, the earlier the onset. Describe experimental evidence, using Southern blotting, that is consistent with this finding.

From the human genome sequence, select _____________ enzymes that will bracket the region of the huntingtin gene that contains the triplet repeat. Obtain DNA from members of families afflicted with Huntington's disease. Carry out Southern blotting with the selected____________ enzymes. Probe each blot with an oligonucleotide (____________ )n or its complement ____________( )n. Or PCR amplify the region of interest and run gel electrophoresis to estimate, from the size of the repeat fragment, the number of repeats; this is what is actually done in HD testing. The age of onset should correlate______________ with the size of the gel band detected by the probe; the larger the band, the ___________ the age of onset.

(Word bank below)

GTC

positively

CAG

restriction

ligation

younger

negatively

older

purplejellyfish572

01:447:384 Study Guide - Final Guide: Southern Blot, Northern Blot, Western Blot

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