The probe attaches in the same location for all three samples. entire size of th

Question

The probe attaches in the same location for all three samples.

entire size of the squence was not given.

For the test, that past obviously, I put D=2kb, Di=3, dii=3

....The "Cut Sites"... Are where the cuts were made after digestion, which happened before southern blotting... she just has the genes still intact for the sake of it being a question.. the ruler is not measuring the size of the probe.. its measuring the length between the two cut sites............................... when is the size of the probe ever measured?

Explanation / Answer

In a southern blot, the DNA to be probed is cut with a restriction enzyme (s), then fragments are separated by gel electrophoresis. Alkali treatment of the gel denatures the DNA which is then blotted onto the filter. A labeled probe (RNA/DNA) is then hybridized to complimentary fragments on the filter.

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

Hybridization of the probe to a specific DNA fragment on filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe. Transfer step of DNA from the electrophoresis gel to a membrane permits easy binding of labeled hybridization probe to size-fractioned DNA. It also allows for fixation of target-probe hybrids, required for analysis by autoradiography or other detection methods. Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine number of sequences (e.g. gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when probe hybridizes to many highly similar sequences. Modification of hybridization conditions (for instance, increasing hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.

Southern transfer may be used for homology-based cloning on basis of amino acid sequence of protein product of target gene. Oligonucleotides are designed that are similar to target sequence. Oligonucleotides are chemically synthesized, radiolabelled and used to screen a DNA library or other collections of cloned DNA fragments. Sequences that hybridize with hybridization probe are further analyzed to obtain full length sequence of targeted gene. Southern blotting can also be used to identify methylated sites in particular genes. Useful are restriction nucleases Mspl and Hpall, both of which brecognize and cleave within same sequence. However, Hpall requires that a C within that site be methylated, whereas Mspl cleaves only DNA unmethylated at that site. Hence, any methylated sites within a sequence analyzed with a particular probe will be cleaved by the former but not the latter enzyme. the size of the probe in measured before fragments are separated by gel electrophoresis.

Related Questions

Navigate

• Browse (All)
• Browse T
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.