Development of the Southern Blotting technique truly revolutionized molecular bi
ID: 3510641 • 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.
Explanation / Answer
The DNA fragment of large size of a particular sequence are possible to be obtained mearly by selective replication. The method for selective replication is called polymerase chain reaction and it uses DNA polymerase and a pair of shorts synthetic oligonucleotides that are complementary in sequence to the ends of the DNA to be amplified and so can serve as primers for strand elongation.
We used the method polymerase chain reaction which produces two copies in the first cycle of each molecule containing copies complementary to the primer.
The second cycle of PCR is similar to the first. After this cycle there are 4 copies of each molecule present in the original mixture.
The different strains of the D. iridis total DNA digested with BamHI are most closely related as we can clearly see in blot there are two copies in third lane.
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