On Tue, 29 Oct 1996, Jeremy Marcus wrote:
> In article
> <Pine.GSO.3.95-960729.961025104728.25836A-100000 at altair.dur.ac.uk>, Malia
> Fullerton <S.M.Fullerton at durham.ac.uk> wrote:
>> : We are having considerable difficulty obtaining readable sequence from
> : several different human nuclear genes. I suspect that GC-richness and/or
> : secondary structure may be influencing sequencing efficiency, as primers
> : placed at different locations give the same poor-quality sequence, and we
> : are using (what has been with other loci) ample quantities of
> : single-stranded template.
> : What are the best (or at least most commonly used) methods for addressing
> : this sort of sequencing problem? References appreciated.
>> Glad you asked. I've been having a lot of trouble with this lately, and
> have tried several methods that have worked with varying degrees of
> success. Here's a synopsis:
>> 1. My original compressions and stops was a reult of using Sequenase 2.0
> to sequence very GC-rich DNA.
>> 2. Inosine reactions using the Sequenase kit didn't seem to work. In
> fact, I've never been able to get them to work. :-/
>> 3. I then tried the Fidelity kit (Oncor), which I saw advertised in
> BioTechniques. Their claim is that, through the use of small DNA-binding
> proteins in the reaction mix, their enzyme will read through structures
> that typically cause stops and compressions. The Fidelity protocol is
> longer and requires more steps than Sequenase, but I figured it was worth
> a shot. Indeed, I got better resolution of many but not all of my stops
> and compressions (70-80% of the problems were eliminated), and the
> protocol was only mildly more tedious than Sequenase's. However, another
> problem erupted: I found that band spacing was not as even as with
> Sequenase, thus raising as many new questions as there were others
> resolved. Other researchers in my lab have said this kit is fantastic,
> and that they'll never go back to Sequenase; I thought it was simply a
> little better than Sequenase. It's pretty certain that you'll get
> improved sequence with this kit, though.
>> 4. By chance I then stumbled across my real salvation: an article in
> BioTechniques 17(2), pages 286-7. This article describes Klenow
> co-sequencing without the extra step of adding the Klenow separately - you
> just add the Klenow to the main reaction mix (along with Sequenase). This
> method worked wonders for me; I got really beautiful sequence, with no
> stops or compressions!
>> Briefly, here's how you do it -
> Proceed as normal with the annealing step, then add 5.5 ul of the
> following mix to the annealed template & primer:
>> (mix recipe for 3 reactions, with a little extra)
> 0.1M DTT 4.0 ul
> undiluted label mix 1.6 ul
> 35S-dATP 2.0 ul
> Klenow 1.2 ul
> H2O 5.2 ul
> Sequenase mix 8.0 ul
>> The Sequenase mix contains:
> 1.0 ul Sequenase
> 0.5 ul pyrophosphatase (included in kit)
> 6.5 ul Sequenase dilution buffer
>> As usual, let the reaction go for 5 minutes at RT, and then proceed with
> the termination reactions at 37 degrees for 30 to 45 minutes (I know that
> sounds like a long time, but believe me, it works!).
>> And yes, there is more glycerol than usual in your reactions due to the
> Klenow and the pyrophosphatase, but don't worry, you probably won't have
> to use glycerol-tolerant conditions. You _may_ need a glycerol-tolerant
> gel for runs that are longer than 4 hours; I can certainly attest to the
> fact that a four hour run looks just fine with a normal TBE gel.
>> Hope this helps! Let me know if you figure out anything else that works better.
I have had the (mis) fortune of having to sequence approx 7 Kb of 70% GC
rich DNA on both strands by hand (14KB!!)
I found a protcol that used DMSO in the reactions and we routinly used it
to get 300bp of good seq. - Basically NaoH denature DNA and HCL neutralise
etc. add 0.5 ul of DMSO ( I have used up to 1ul) ro annealing step - then
0.68 ul to extension step and 0.25 ul to each of the terminators. Running
the gel at 55 C and doing the reactions at 42 - and using deaza mixes was
also used with the DMSO.
If u want more specific details as I'm not sure about the amounts
specified about (its been a while!) then just mail me and I'll send you
I.G.Goodfellow at reading.ac.uk