Development of the Southern Blotting technique truly revolutionized molecular bi
ID: 199606 • Letter: D
Question
Development of the Southern Blotting technique truly revolutionized molecular biology and has led to the development of several related techniques based on the same principle. A probe molecule is selected and labeled, then, through the hybridization process, we are asking if any of the DNA samples on the gel (which were transferred to the blot) have the same or closely related DNA sequences as our probe. Because the gel has fractionated the DNA based on the fragments sizes generated by restriction enzyme digestion we can learn something about the size of the fragments showing similarity to our probe. Last week you made a Southern blot of digests of Didymium iridis total DNA that you isolated. After allowing the transfer to proceed for two hours the blots were rinsed in 2 x SSC (Saline, Sodium Citrate) to remove precipitated salt and bits of agar. UV light was used to cross-link the DNA to the membrane. Then pre-hybridization of the blots was carried out in the following solution: 5 x SSC, 0.5% milk protein, 0.1% sarkosyl (a detergent), and 0.02% SDS (another detergent) at 42°C for 1 hour. At the end of the pre-hybridization reaction, the pre-hybridization buffer was replaced with a smaller volume of hybridization solution, the same buffer as above, but also containing your labeled probe DNA which was heat denatured for 10min at 100°C, then quick cooled. The hybridization reaction was allowed to proceed overnight. The probe can be saved and re-used for a couple of more blots. The blots were then rinsed free of unbound probe by washing with solutions of SSC and 0.1% SDS at 42°C. The hybridization membranes were then air-dried.
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1. Suppose the fragments of DNA you labeled to make your probe was a 2.5 kb EcoRI restriction fragment of Didymium strain Pan 2-16 IDNA. You hybridized the probe to a blot of BamHI digested total DNA from a variety of Didymium strains. Your results are pictured below. Blot Gel Lane 1 -HindIII marker DNA Lane 22.5 kb EcoRI fragment Lanes 3-5 Different strains of D. iridis total DNA Digested with BamHI Probed with EcoRI-2.5 kb fragment a) Explain why the probe hybridizes to fragments larger than itself. b) Give a reasonable explanation for the observation that the probe hybridizes to two bands in the strains in the last lane (assume gene duplication has not occuired) c) If you were trying to use this data to infer which strains are most closely related, what would you conclude? Give your reasoningExplanation / Answer
a) in this experimet as a probe a 2.5 Kb EcoRI digested fragment were used. The genomic DNA D.iridis was digested with BamHI. In the blot the band hybridized with EcoRI appears lager. This could happened as EcoRI and BamH1 has different resriction site in the genomic DNA. Thus BamHI cuts the DNA on different site in comparison to EcoRI and generaed fragments which is larger than 2.5. However, as 2.5 Kb long DNA sequence is hybridizing with the complimentary sequence which falls wihtin the BamHI fragment. At the end the the band legth appears larger than the probe length.
b) If BamHI has an additional restriction site within the 2.5 Kb region of EcoRI digested fragment, the EcoRI digested probe can hybridize both the fragments and there will be two bands on the blots and will be smaller in size than the EcoRI probe.
c) Strain 1 and 2 are more closesly related to each other, as they have same BaHI restriction site, whereas in strain 3, the BamHI restriction sites are different.
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