David111 Posted February 27, 2015 Share Posted February 27, 2015 (edited) My teacher has given this question that nobody in the class seems to understand. Can anybody point me in the right direction? Q. You wish to know whether the cDNA you have isolated and sequenced is the product of a unique gene or is made by a gene that is a member of a family of related genes. To address this questions, you digest cell DNA with a restriction nuclease that cleaves the genomic DNA but not the cDNA, separate the fragments by gel electrophoresis, and visualize bands using radioactive cDNA as a probe. The Southern blot shows two bands, one of which hybridizes more strongly to the probe substance than the other. You interpret the stronger hybridizing band as the gene that encodes your cDNA and the weaker band as a related gene. When you explain your result to your advisor, she cautions that you have not proven that there are two genes. She suggests that you repeat the Southern blot in duplicate, probing one with a radioactive segment from the 5'-end of the cDNA and the other with a radioactive segment from the 3'-end of the cDNA A) How might you get two hybridizing bands if the cDNA was the product of a unique gene? B) What results would you expect from the experiment your advisor proposed if there were a single unique gene? Or if there were two related genes? Thanks in advance to any geniuses that can lend me a hand. Edited February 27, 2015 by David111 Link to comment Share on other sites More sharing options...
fiveworlds Posted February 27, 2015 Share Posted February 27, 2015 (edited) You wish to know whether the cDNA you have isolated and sequenced is the product of a unique gene or is made by a gene that is a member of a family of related genes As in genetically related?? Place the test subject(unknown child) and father dna side by side and visualize the bands. If the child is related it will have dna markers from the father. This is assuming that the mother being the mother of the child isn't in doubt. If you wanted to be really specific about it you could test the mother too to ensure that there isn't a stray band anywhere She suggests that you repeat the Southern blot in duplicate, probing one with a radioactive segment from the 5'-end of the cDNA and the other with a radioactive segment from the 3'-end of the cDNA Your radioactive segment probe will only test for one segment at a time therefore the southern blot has to be duplicated because some bands can appear similar with respect to electrophoresis but under more stringent tests are not similar at all also the southern blot involves bathing the sample in each radioactive probe Edited February 27, 2015 by fiveworlds Link to comment Share on other sites More sharing options...
David111 Posted February 27, 2015 Author Share Posted February 27, 2015 I'm not sure this is a test between two strands of human DNA. It's kind of just a generic specimen. Not trying to determine parenthood. Link to comment Share on other sites More sharing options...
fiveworlds Posted February 27, 2015 Share Posted February 27, 2015 I'm not sure this is a test between two strands of human DNA. It's kind of just a generic specimen. Not trying to determine parenthood. You have to know whether the cDNA you have isolated and sequenced is the product of a unique gene or is made by a gene that is a member of a family of related genes. Not necessarily human DNA but parenthood all the same Link to comment Share on other sites More sharing options...
Robittybob1 Posted February 27, 2015 Share Posted February 27, 2015 I'm not sure this is a test between two strands of human DNA. It's kind of just a generic specimen. Not trying to determine parenthood. I'm surprised at the level of education in your country. What age group are you may I ask? Link to comment Share on other sites More sharing options...
fiveworlds Posted February 28, 2015 Share Posted February 28, 2015 I am 21. However genetics is not my specialty I just wanted a challenge Link to comment Share on other sites More sharing options...
Robittybob1 Posted February 28, 2015 Share Posted February 28, 2015 I am 21. However genetics is not my specialty I just wanted a challenge I wonder what level David111 is at for that seems a pretty complex question. Link to comment Share on other sites More sharing options...
CharonY Posted February 28, 2015 Share Posted February 28, 2015 (edited) Actually it is not. But it is more of a technical kind, but there is one thing that I find not precisely expressed. What you need to know what cDNA is and how it is produced. Based on that It is weird that it states that the restriction enzyme does not cleave the cDNA when you digest the genomic DNA. Can you tell me why? Hint: it probably means that the restriction site is not found in the cDNA sequence. Revisit how the cDNA may differ from the actual genomic sequence. Based on that, what are potential causes of multiple bands to which the probe hybridize to? Why would labeling the different end make any difference? It should be noted that the interpretation may be different if you have a pro- or an eukaryote. I assume it is the latter, though. (Also, not to be mean, but ignore the stuff above it is incorrect and has no bearing on the question whatsoever). Also I am going to move it to the homework section. Edited February 28, 2015 by CharonY 1 Link to comment Share on other sites More sharing options...
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