sothing about Genome walking

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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hi ,all

I want to ask that the Tm's of AP1 and AP2 are 59¡ãC and 71¡ãC,which is written in the USER MANUAL,but I get 51.1¡ãC and 57.9¡ãC in the software of PRIMER PREMIER 5.0. If I use different softwares I will get different TM values, but they are all lower than those in the USER MANUAL. So whether I should correspondly low the special primers(GSP)'s TM values( recommended >=67¡ãC), when I design them.How do you deal with them .Thank you!

-edss-

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what I said above is all about the Universal genomewalker kit

-edss-

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edss-

Yes, you will get different Tm estimates for primers using different software, as each uses a slightly different formula to calculate the Tm. I use the Genomewalker kit. I haven't figured out why the Tm of AP1 is so low when they say to design your genespecific primers with a Tm of 70... However, the best way to determine the annealing temperatures to use is trial and error. If you have access to a gradient thermocycler, I'd run a test on one or two samples using annealing temperatures from below 60 to 72.

I have found that an annealing temp of 65 works well with my samples and genespecific primers. I am using Expand polymerase systems from Roche, which preferentially extend at 68, and peltier-device thermocyclers from MJ Research. BTW, I work on grass. I have also found that I can get the human DNA supplied as a control with the kit to successfully amplify under conditions which won't amplify the grass DNA.

Hope this helps.

Turtle

-turtle-

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Turtle :

Thanks for your useful help! I will try it as you said .

And I also have a question:

when you design the two nested primers GSP1(genespecific primer)and GSP2, what is the best distance from the GSP2 to the 5'end of my DNAsequence(when i want to upstream) ?And what's the best distance between the two nested primers ?

Thanks for a lot!!!

-edss-

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QUOTE

when you design the two nested primers GSP1(gene specific primer) and GSP2, what is the best distance from the GSP2 to the 5' end of my DNA sequence (when i want to upstream) ? And what's the best distance between the two nested primers ?

I don't think there is a "best", but there are points you should take into consideration.

For the distance between GSP2 and the known 5' end of your sequence, don't put GSP2 at the most 5' end of known sequence because if you get PCR product with GSP2 and have sequenced it, you have to check if the new sequence overlaps the 5' end of known sequence.

The distance between GSP1 and GSP2 is not important. They can even overlap.

-mario2004-

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mario2004:D

Thank you very much for your help!!!

edss

-edss-

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Mario is right, the distance isn't important. I generally try to design my GSPs far enough 3' that I can also squeeze in a sequencing primer 5' of te nested GSP for walking upstream. I direct sequence my products, and find that having the sequencing primer nested inside the GSP works better than just using the nested GSP as a sequencing primer.

Turtle

-turtle-

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thank you for your help!

may i ask which software do you use to decide the TM values of the primers?

-edss-

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It's already March... but I just wanted to briefly mention about my experience with Seegene's DNA Walking SpeedUp Kit that kiwi mentioned. I had a friend who tried it to do BAC cloning and she recommended it to me.

I had a piece of known seuqence and wanted to find out the sequence in the unknown region.

It was very easy to use and I had about 1Kb for each walking. I think the advantage of using this kit is that you only get real products (save so much time).

However, you have to design 3 target specific primers(TSPs) for nested PCR and it's quite crucial to have good TSPs for good result.

If you do try, don't forget to complete 3 PCR reactions because I didn't get any bands until the 3rd PCR.

They have a small pack.. 10 reaction.. that's what I first tried..

-without00-

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[i hope you could elaborate on the TSPs... I am also interested of making use of seegene... how much did it cost? thanks!

-Sequencer-

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QUOTE(Sequencer @ Mar 22 2005, 12:47 AM)

[i hope you could elaborate on the TSPs... I am also interested of making use of seegene... how much did it cost? thanks!

[snapback]12708[/snapback]

TSPs are just primers you will use to amplify unknow DNA sequence with a link primer in genome walking. You can design it by yourself and order it from any company.

-pcrman-

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QUOTE(pcrman @ Mar 22 2005, 07:41 PM)

TSPs are just primers you will use to amplify unknow DNA sequence with a link primer in genome walking. You can design it by yourself and order it from any company.

[snapback]12766[/snapback]

i aslo sent this messeage to without00.

i am using seegene kit, but i've got nonspecific sequence. it is amplified by ACP primers. so i am wondering how too design an effective TSP primer.

according to the mannual, ACP primer-binding sites(5-XGGTC-3) upstream of TSP1 or in/between TSP primers should be avoided. why is this a big deal? there must be a limit of distance for the distance. i mean we should only consider a certain distance upstream, like within 1kb. am i right?

how about those binding sites downstream of the TSP primers? doesn't matter?

how long fragment can you get using this kit? can we use long PCR system instead.

hope you can share your expereince with us.

thanks a lot.

-seqman-

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Hi all,

I have recently used Clonetech's Universal genome walker kit with pretty good results.  I did have to do a bit of tweeking though. 

First of all, the annealing Temps. suggested in the kit didn't work, not on the controls or my specific samples.  I went to gradient PCR to figure out the best temp for each combination of primers (adapter & specific).  Next, after doing all of this trouble-shooting, I was out of Taq and didn't want to spend an enormous amount of money to replace their proprietary Taq, so I tested a few and found that Invitrogen's Platinum Hi Fidelity Taq worked well at about 1/6 the cost of their Taq. 

As for cloning vs. direct sequencing, I never direct sequence unless I'm > 90% sure I'm only amplifying one product.  When walking through a 'library', there is a high chance that you will get more than one product in any PCR reaction or gel purified band for that matter.  I use pGEM-T easy, love it, have cloned larger inserts (2.5 kb from this walk) and it's easy to sequence using standard primers.

My organism is ryegrass and I've obtained additional 5' and 3' sequences, but still not to the ends of the gene.  The trouble is that there are introns in my genomic DNA, so now I'll resort to RACE!

-grassgirl-

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hello segman,

i am sorry for this late reply.

I think the theory behind Seegene's DNA Walking kit is pretty complicated... from what I understand, TSP1 is crucial. They employed ACP technology in this kit, which is supposed to maximize PCR specificity. Therefore, as long as the products from the first PCR is specific, you will continue to get specific products in the following PCR reactions(nested PCR reactions).

if you have got non-specific bands, i think you should write to seegene. they have some sort of back-up sheet for technical support... my friend needed some technical support for DNA Walking kit and she resolved her problem that way.

She was told that the non-specific bands from ACP primers was created because there was no target product created during the first PCR reaction. She, then, had to change her TSP 1 in the second trial. The job of ACP primer in this kit is to randomly bind to any XGGTC site in your unknown region. That's why you should avoid this sequence between TSPs.

I got a squence upto about 1.3 Kb. It really depends on the chance of XGGTC appearance in your template.. I mean, mathmatically XGGTC should exist in every 1Kb, but it depends on your template.

I also heard that ACP doesn't work with enzymes that has a proofreading activity. This enzyme is supposed to interfere the activity of DW-ACP primer.

oh, by the way, this is for sequencer...

TSP is target specific sequences. You have to design 3 TSPs in the region with known sequences for nested primer.

10 reaction kit was about USD240. i am not sure what it costs outside of US though.. you can download the manuals from their website. they mention criteria for TSP design in the manual... hope this helps!

-without00-

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Hi

Just for your information,

i have used Seegene's DNA Walking SpeedUp Kit before. erm... with the ACP primer technology in the kit, we managed to get the promoter region of the gene.

After the third round PCR, we sent the amplicon for direct sequencing without cloning. The result was ok.

good luck and all the best...

-yeanbg-

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How does this approach compare to inverse-PCR? It seems to me that inverse PCR is more straightforward, and requires only standard reagents, a little cleverness, and far less optimization.

-phage434-

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Hi there

I have been using TOPO TA cloning kit routinely for cloning and then sequencing. i have always felt confortable with M13 preimers for sequencing. If your product size is really big i mean more than 4-5k then go for TOPO XL. you can use XL up 35-40K with great efficiency.

Good luck

QUOTE(kiwi @ Sep 8 2004, 08:20 PM) [snapback]7022[/snapback]

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

-Mikroman-

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can you tell me what is genome walking.........

regards

amit

-Amit Kumar-

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QUOTE(Amit Kumar @ Dec 5 2005, 01:22 PM) [snapback]33448[/snapback]

can you tell me what is genome walking.........

regards

amit

Hi

Have a look at this

Devic et al., Plant Physiol. Biochem, 1997, 35 (4)

I have been in this lab' to learn the technic but I have never used Clontech's kit. I find it's a waste of money.

You can just make the AP1, AP2, and GSPs and buy the whole lot separately. Saves you some cash.

It always worked fine in my hands but it is important to have at least 5 different "libraries" because you don't always have results with all of them.

The use of a gradient thermocycler seems to bee a good idea too..

-BillyBoy-

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I have used the Universal BD Genome walker kit from CLONTECHfor genome walking in plants.It worked well but got only a700 bp fragment upstream

-Indian-

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QUOTE(Amit Kumar @ Dec 5 2005, 05:52 PM) [snapback]33448[/snapback]

can you tell me what is genome walking.........

regards

amit

HI Amit

It is a method to isolate the sequences upstream of a known sequence.This is usually done to isolate promoters or flanking sequences of a gene. Refer Clontech's Genome Walker kit' user Manual

-Indian-

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I have used the seegen' s kit in fungal experiments, it is easy to use, and well performed.

-~M~LLM~-

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QUOTE(kiwi @ Sep 2 2004, 06:13 AM) [snapback]6918[/snapback]

Hi all,

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!

Hi

Is there any company other then clontech which offer genome walking kit or related technique to find out unknown sequences from DNA.?

regards

-asmi-

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Hi everyone,

I have some problems with primer design in my Genome Walker experiments. I decided to use Universal Genome Walker Kit (CLONTECH). The problem is with Tm of primers. As sb said earlier different programs give you diffrent Tm. Can anyone tell me what is the best method used by program to assume Tm? Do I have to take into account NN, basic or salt adjusted (nearest neighbour, 2AT+4GC and %GC respectively)?

Thanks very much for help:)

-Diamanda-

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hi ,everyone !

i have used the Universal BD Genome walker kit from CLONTECHfor genome walking in plants. But it didn't work very well. i always got a band of 5~6kb, however when cloned into pmd-t vector, it was only 2kb.

I don't know what is wrong and how to deal with it ? Can any one help me ?

-danshen-

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QUOTE(danshen @ Nov 8 2006, 08:18 AM) [snapback]76174[/snapback]

hi ,everyone !

i have used the Universal BD Genome walker kit from CLONTECHfor genome walking in plants. But it didn't work very well. i always got a band of 5~6kb, however when cloned into pmd-t vector, it was only 2kb.

I don't know what is wrong and how to deal with it ? Can any one help me ?

I just wanted to say that I am experiencing the same problem. I am working with plant DNA as well (specifically potato) and I have isolated a ~2kb band and gel purified it and then cloned the purified product into pGEM-T easy vector and when I did PCR on the subsequent colonies it indicated that I only had ~400bp.

Although, I am not able to help in this situation I wanted you to know that you are not the only one experiencing it. If you figure it out let me know and I will do the same.

-potato girl-

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I also have been running into problems with the Universal genome walker kit. I am trying to clone a promoter from Chinese chestnut and had to design my primers from a partial known sequence.

My first walk produced bands at 3 kb, ~1750kb, and smaller sized fragments. Upon cloning (TA cloning kit), however, only the smaller fragments were successful. At first the PCR protocol was not successful, -  - my GSP1 had a Tm of 71 & GSP2 had a Tm of 70. I had to redesign my GSP1 with a new Tm of 61 and then use a 3-step pcr program.

I am now conducting a second walk in both directions with new GSP's. Is it necessary to conduct both the primary and secondary pcr again, or should I try a pcr with my new GSP & AP2?

Does anyone have optimizations for using a mj research ptc-150 minicycler?

Thanks

Biotech Stumpie

-stumpie-

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I am doing genome walking to fish the promoter sequences of my gene by using PCR LA kit (TaKaRa). I performed restriction cuttiing with six enzymes and ligated with the casette provided by the kit. When I did PCR with my specific primer of known region and casette primer. It had only short PCR product 400 bp obtained from Sau3AI cut chromsomal fragments. Can anyone give me the suggestion why I obtained only short PCR fragment and how can I solved the problem to get the long PCR fragment. By using this kit how can I decided with restiction enzyme to be chosen to cut the chromosome before doing genome walking. The kit has EcoRI, HindIII, PstI, SalI, Sau3AI and XbaI cassettes.

Thank you

-Thai-

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I am trying to walk a 9 Kb gene, but so far I have no success in getting fragment. I always get 300 to 500bp fragment. Is it my primers doing this or is there any other way that ican obtain a bigger frament.

please help

-juliegeorge-

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If your gene is cloned and is only 9Kb, then you can sequence it directly without attempting to subclone it. Sequence in from both ends as far as possible. Choose two primers near the end of reliable sequence from those read, and sequence forward again. At the same time, choose two reverse primers from the very reliable middle of the sequence read and sequence those for verification. You can advance about 800 bp at each end per round, or about 1600 bp, so in about six rounds you will have the complete sequence. You can speed this up even more if you know of a starting sequence in the middle, of course. This is all far easier and more straightforward than the complex "kit" protocols of chewing back ends, partial cutting with enzymes, subcloning, transposon insertion, whatever.

-phage434-

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QUOTE(phage434 @ Aug 10 2007, 07:48 AM) [snapback]107986[/snapback]

If your gene is cloned and is only 9Kb, then you can sequence it directly without attempting to subclone it. Sequence in from both ends as far as possible. Choose two primers near the end of reliable sequence from those read, and sequence forward again. At the same time, choose two reverse primers from the very reliable middle of the sequence read and sequence those for verification. You can advance about 800 bp at each end per round, or about 1600 bp, so in about six rounds you will have the complete sequence. You can speed this up even more if you know of a starting sequence in the middle, of course. This is all far easier and more straightforward than the complex "kit" protocols of chewing back ends, partial cutting with enzymes, subcloning, transposon insertion, whatever.

I am sorry my question was not quite clear This 9kb gene is the gene of Arabidopsis and I am trying to get the same gene in Eucalyptus sp. So I am using the genome walking procedure to get the same gene in my species. So when I use the genome walking kit I only get 300 bp fragment. I am using 8 different libraries. But no success in getting larger fragments so far. Please help

-juliegeorge-