Could I have some assistance in regards to Hybridisation.

[sillysmile]

Okay, I was asked by my heinemann biology textbook (in Key questions 11.1)
'Explain what Hybridisation means in molecular biology.'
along with
'Explain how hybridisation is used in this technique to find a particular sequence of DNA.'
I could not understand the answer these questions, could somebody please explain what the answer should be, to me.
+1 karma is at the disposal of anybody who helps

[Russ]

Hybridisation is the process of joining (annealing) two pieces of single stranded DNA. If you mix two pieces of ssDNA that have complementary sequences, the bases will pair up and it becomes double stranded DNA. So, if we know what the sequence is of one of the pieces of ssDNA, we can therefore locate the complementary sequence on the other piece (because it will ONLY bind and form dsDNA if the exact sequence is already present).

Say you have a bacterial chromosome and you're investigating a gene to find out if it contains a mutation. You produce (typically via PCR) lots of copies of the gene, which you can then denature and make single stranded and plate. Then, if you add pieces of ssDNA that are complementary to the mutation under investigation two things can happen: if the gene DOES have the mutation, the DNA will anneal and you'll see dsDNA. If all the DNA remains single stranded, you can conclude the gene does not have that particular mutation.

Hope those covered your questions, I realised I didn't actually address them properly
If you have anything else, i'll give it a shot!

[sillysmile]

So hybridisation is the joining of complementary DNA strands?

thank you Russ, that actually was a lot simpler then I believed, I also realised that I left out that the questions were relating to southern blot hybridisation. You enabled me to understand the concept of hybridisation

Also one other question.. A primer is a short sequence of RNA that is bound to ssDNA??
haha, I don't really understand the concept of Primers, could you please elaborate?

[Russ]

Southern Blot is just one of a whole bunch of different specific lab techniques, the general principle is the same.

Primers allow DNA replication because DNA polymerase can't start building a new strand by itself. So a primer binds to the ssDNA and allows DNA polymerase to synthesize the complementary strand by continuing on from where the primer ends (the primer is later removed by a different polymerase and the gap sealed with ligase). In my last post I mentioned how you'd use PCR to produce lots of copies of the DNA sequence you wanted to investigate...well, you need to add primers along with the ssDNA in order to make all those extra copies.

e, yeah primers are RNA sequences that bind to ssDNA (in almost all cases)

[sillysmile]

Yeah, that all makes crystal clear sense, thank you . One other thing, Are primers added to the end of a sequence also?

[happyhappyland]

Yeah, that all makes crystal clear sense, thank you . One other thing, Are primers added to the end of a sequence also?

No, I think primers are only added at the 5' end.

[Russ]

5' end of the ssDNA? If the primer attaches there it won't be able to build anything. In DNA replication, it does this by Okazaki fragments, but for an isolated piece of DNA there's nothing to build off.

[happyhappyland]

5' end of the ssDNA? If the primer attaches there it won't be able to build anything. In DNA replication, it does this by Okazaki fragments, but for an isolated piece of DNA there's nothing to build off.

The primer itself has a 3' OH end and DNA polymerase builds from 5' to 3' ends. Does that make more sense? So sort of like this


........::::::......... Bases added this way ---->>>>

The right side of the ::::: is the 5' end of the new strand (of the top dots)

[Russ]

The primer itself has a 3' OH end and DNA polymerase builds from 5' to 3' ends.

Yes, but then you've said the opposite with your diagram. If the right hand side is the 5' end of the new strand, then you can't build in that direction after attaching the primer; you can only add new bases to the left of the top strand, to the 3' end.