sothingaboutGenomewalking

Can anyone recommend a good kit for genome walking? I would like to find out the promoter sequence of a known gene. Is genome walking the way to do it?

thanks a million for any suggestions!

-kiwi-

Kiwi-

I use the Universal GenomeWalker Kit from BD Biosciences. The kit includes restriction enzymes to cut the DNA and adapters to ligate to the DNA, as well as primers specific to the adapters. It does not include the polymerase, although you can buy a supposedly optimized polymerase from BD.

The kit works OK, and is not too expensive. The one difficulty I had is finding a polymerase which worked on my templates. The controls with the kit (human DNA) worked fine with all the high-fidelity polymerases I tried, but most wouldn't work on my plant DNA. I settled on the Extend High Fidelity Polymerase system from Roche, and the Phusion polymerase from MJ Research.

Otherwise, I was somewhat disappointed in that the kit, which hasn't been updated recently, doesn't really seem to take advantage of the long amplification distances possible with the newest polymerases. Most of my amplicons were 3 kb or less; the longer ones were all amplified from both ends by the adapter primers, rather than my gene-specific primer. I don't know if there are other kits out there that make better use of the current technology.

Hope this helps.

Turtle

-turtle-

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Hi Turtle,

Thanks for the information. I am still a bit clue-less. I am quite new to this technique...After i use the enzymes from the kit and get the bands that i want, do i clone them and sequence them using the same primers that i designed for the initial PCR reaction? Which vector should i use? Do you have any to recommend?

Also, have you heard of Seegene's DNA Walking SpeedUp™ Premix Kit? I saw it on the net. Like all products, they claim that they are good.

Thanks once again!!

Kiwi

-kiwi-

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Kiwi-

I sequenced my bands directly, without cloning. I have a beastly time with cloning larger (>500 bp) fragments of ryegrass DNA, so I try to avoid doing so

I have used the nested gene-specific primer for sequencing the GenomeWalker fragments; it works OK but you have to be very careful not to degrade the 5' end of your fragment or the primer won't bind. Primers are relatively inexpensive, so I often nest a third primer inside my nested gene-specific primer and use that for sequencing. Theoretically I suppose you could use the nested adapter primer for sequencing also, although I have never tried this.

I've never heard of the Seegene kit. BTW, what species are you working on?

-Turtle

-turtle-

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Hi Kiwi,

Why don't you try a sucide plasmid directed mutagenesis. As your gene is known, you can clone it (or a part of it) into a plasmid that will not replicate in your strain. So, you are going to do a site specific recombination. After cutting the DNA with enzymes that do not cut your plasmid you are able to recover the sequence flanking the insertion site. Now you have your region of interest on a plasmid.

-Sphingoman-

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Hi Kiwi,

Which organism are you working on? If possible, try to get the sequence from database, such as ensembl, ucsc genomic database.

Many years ago I used Clontech's genome walking kit and had successfully cloned a promoter region which is GC rich. I think they are (now BD) still selling that kit.

QUOTE

do i clone them and sequence them using the same primers that i designed for the initial PCR reaction? Which vector should i use? Do you have any to recommend?

You can do a direct sequencing using both primers used for PCR. If you do want to clone it, just do a TA cloning. I recommend Invitrogen's Topo TA cloning kit.

-pcrman-

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Hi all,

Me working on mouse and human genes now. I used to work on plants so now am a bit phobic on starting on something new.

I heard before (during my plant days ) that the TOPO TA cloning kit is good. Is there a limit to the size of the insert that it can take? I was told to clone at least 2kb insert.

On the other hand, direct sequencing seems like a good idea although i have some friends who tell me its impossibly difficult to direct sequence. Is that true?

I just saw a splinkerette method on the net....has anyone tried that before??

To Sphingoman:

Do you have any good reference or article for me to read on the sucide plasmid directed mutagenesis method?

Thanks a million for all the input!

kiwi

-kiwi-

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2 kb is OK with TA cloning. I don't see any difficulties in direct sequencing. The only problem is you may not get the full sequence by two (using upstream and downstream primer) sequencing reaction. In that case, you have to design a new primer on the newly obtained sequence to sequence the rest of it. But this problem also applies to sequencing after cloning.

-pcrman-

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I have another query:

I am working on the mouse genome and since the entire mouse genome is known, is it better to 1) genome walk in order to "fish out" the promoter sequence or 2) to design primers and run a normal PCR to "fish out" the promoter?

If I use method 2), is there a limit to the length of the PCR product that i can obtain using Taq polymerase available in the market? Which brand/kind of Taq would be recommended?

blur,

kiwi

-kiwi-

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In that case, I don't think you need to do a genome walk to find the upstream sequence since it is already known. If you want to clone it for the characterization of the promoter sequence, just do a PCR cloning. Certainly there is a limit in product size a taq can amplify but usually what a taq can amplify (for example, several kb) is sufficient for your purpose. There are DNA polymerases specifically made for long-distance PCR or high fidelity PCR.

-pcrman-

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