Genome walking - Urgent help! - Genome walking (Jul/15/2008 )
Hello all. I'm a student undertaking a diploma course in Molecular Biotechnology. I do need to seek help regarding the Genome walking method. Of particular interest is the protocol for the GenomeWalker kit by Clontech.
I do know that Genome walking is used to identify unknown flanking regions of a known region. I have read the Clontech GenomeWalker manual several times, but I still do not understand how the method works. In particular, where does AP1/AP2 and GSP1/GSP2 anneal to, and how does this method eventually lead to the identification of unknown flanking regions.
I am supposed to prepare a presentation based on a journal paper and in the journal paper, the authors had used genome walking as a method to obtain complete ORFs, as well as upstream and downstream sequences of several unique fragments derived from suppressive subtractive hybridization. They were investigating the differences in genome between 2 strains of the same bacteria.
Any help would be much appreciated. Thank you for reading this!
-Sdyter-
QUOTE (Sdyter @ Jul 15 2008, 03:00 AM)
Hello all. I'm a student undertaking a diploma course in Molecular Biotechnology. I do need to seek help regarding the Genome walking method. Of particular interest is the protocol for the GenomeWalker kit by Clontech.
I do know that Genome walking is used to identify unknown flanking regions of a known region. I have read the Clontech GenomeWalker manual several times, but I still do not understand how the method works. In particular, where does AP1/AP2 and GSP1/GSP2 anneal to, and how does this method eventually lead to the identification of unknown flanking regions.
I am supposed to prepare a presentation based on a journal paper and in the journal paper, the authors had used genome walking as a method to obtain complete ORFs, as well as upstream and downstream sequences of several unique fragments derived from suppressive subtractive hybridization. They were investigating the differences in genome between 2 strains of the same bacteria.
Any help would be much appreciated. Thank you for reading this!
I do know that Genome walking is used to identify unknown flanking regions of a known region. I have read the Clontech GenomeWalker manual several times, but I still do not understand how the method works. In particular, where does AP1/AP2 and GSP1/GSP2 anneal to, and how does this method eventually lead to the identification of unknown flanking regions.
I am supposed to prepare a presentation based on a journal paper and in the journal paper, the authors had used genome walking as a method to obtain complete ORFs, as well as upstream and downstream sequences of several unique fragments derived from suppressive subtractive hybridization. They were investigating the differences in genome between 2 strains of the same bacteria.
Any help would be much appreciated. Thank you for reading this!
Never used the Genomewalker kit myself, but I looked at the protocol and will try to explain it to you.
So you start by isolating your genomic DNA and you cut it with a blunt end restriction enzyme (they make you do 4 separate reactions). This will generate many small pieces of DNA (your library), some of which will contain your known region of DNA that also have adjacent unknown DNA as well. For example, you might generate a 3 kb piece and the right 1/3 (or 1 kb) of it is your known sequence, but the rest of it (2 kb) you don't know. Now you need to determine what the sequence is of this unknown 2 kb sequence is. To figure that out you need to be able to make PCR products for it, but obviously you can't because you don't know any sequence to make a left flanking primer. This is where the kit comes in. The kit provides linker sequences (the Genomewalker adaptor in the figure) that you ligate to your 3 kb piece using a ligase enzyme. Since this linker sequence is known, you can now do PCR. You use AP1 (adaptor primer 1) and GSP1 (your gene specific primer 1 - a primer that you design from your known sequence) to do the PCR as shown in the figure in the manual. They show this as your first PCR product. It's possible that this would be good enough to get sequence from it, but to make it even cleaner they do nested PCR to get really good sequence. This is where AP2 and GSP2 come in. You use the first PCR product and these primers, which are located further inside the first PCR product, to get a second PCR product which you can then clone and sequence.
Now lets say that you do this sequencing reaction, and you get some new sequence that you previously didn't know. You can take this new sequence and repeat the process. Somewhere in the library that you generated is another piece of DNA which has this new sequence and more flanking unknown sequence. Make a new GSP1 and GSP2 primer for the sequence you just discovered and perform the PCR again. You continue to repeat this until you either run into something that was previously known or you run into the end of the chromosome. In this way, you can "walk" along the chromosome and fill in regions of DNA where the sequence was previously unknown.
Hope this explanation makes sense and is helpful to you.
smu
-smu2-
QUOTE (smu2 @ Jul 15 2008, 10:22 PM)
QUOTE (Sdyter @ Jul 15 2008, 03:00 AM)
Hello all. I'm a student undertaking a diploma course in Molecular Biotechnology. I do need to seek help regarding the Genome walking method. Of particular interest is the protocol for the GenomeWalker kit by Clontech.
I do know that Genome walking is used to identify unknown flanking regions of a known region. I have read the Clontech GenomeWalker manual several times, but I still do not understand how the method works. In particular, where does AP1/AP2 and GSP1/GSP2 anneal to, and how does this method eventually lead to the identification of unknown flanking regions.
I am supposed to prepare a presentation based on a journal paper and in the journal paper, the authors had used genome walking as a method to obtain complete ORFs, as well as upstream and downstream sequences of several unique fragments derived from suppressive subtractive hybridization. They were investigating the differences in genome between 2 strains of the same bacteria.
Any help would be much appreciated. Thank you for reading this!
I do know that Genome walking is used to identify unknown flanking regions of a known region. I have read the Clontech GenomeWalker manual several times, but I still do not understand how the method works. In particular, where does AP1/AP2 and GSP1/GSP2 anneal to, and how does this method eventually lead to the identification of unknown flanking regions.
I am supposed to prepare a presentation based on a journal paper and in the journal paper, the authors had used genome walking as a method to obtain complete ORFs, as well as upstream and downstream sequences of several unique fragments derived from suppressive subtractive hybridization. They were investigating the differences in genome between 2 strains of the same bacteria.
Any help would be much appreciated. Thank you for reading this!
Never used the Genomewalker kit myself, but I looked at the protocol and will try to explain it to you.
So you start by isolating your genomic DNA and you cut it with a blunt end restriction enzyme (they make you do 4 separate reactions). This will generate many small pieces of DNA (your library), some of which will contain your known region of DNA that also have adjacent unknown DNA as well. For example, you might generate a 3 kb piece and the right 1/3 (or 1 kb) of it is your known sequence, but the rest of it (2 kb) you don't know. Now you need to determine what the sequence is of this unknown 2 kb sequence is. To figure that out you need to be able to make PCR products for it, but obviously you can't because you don't know any sequence to make a left flanking primer. This is where the kit comes in. The kit provides linker sequences (the Genomewalker adaptor in the figure) that you ligate to your 3 kb piece using a ligase enzyme. Since this linker sequence is known, you can now do PCR. You use AP1 (adaptor primer 1) and GSP1 (your gene specific primer 1 - a primer that you design from your known sequence) to do the PCR as shown in the figure in the manual. They show this as your first PCR product. It's possible that this would be good enough to get sequence from it, but to make it even cleaner they do nested PCR to get really good sequence. This is where AP2 and GSP2 come in. You use the first PCR product and these primers, which are located further inside the first PCR product, to get a second PCR product which you can then clone and sequence.
Now lets say that you do this sequencing reaction, and you get some new sequence that you previously didn't know. You can take this new sequence and repeat the process. Somewhere in the library that you generated is another piece of DNA which has this new sequence and more flanking unknown sequence. Make a new GSP1 and GSP2 primer for the sequence you just discovered and perform the PCR again. You continue to repeat this until you either run into something that was previously known or you run into the end of the chromosome. In this way, you can "walk" along the chromosome and fill in regions of DNA where the sequence was previously unknown.
Hope this explanation makes sense and is helpful to you.
smu
Hello. Thank you so much for your help! Your explanation was crystal clear. Now I get a clear picture of how genome walking is done..thank you for spending time to read the protocol..as well as replying to my query. Thank you!
-Sdyter-
No problem. Glad to help.
smu
-smu2-