まだ途中経過ですが。
A1さんのアドバイスと他に何か方法がないかを委託先に相談してみたところ以下のような回答が
ありました。こちらから依頼する前に既に彼らの方でいくつかの条件を検討した結果、二次構造の
問題と結論したようです。何とかするしかないのですがその道はまだ長そうです。
Dear Dr. 二次構造,
The signal drop might be caused by the presence of secondary structures in
the DNA. In your case, the results obtained with the forward and reverse
primers suggest the presence of a strong hairpin.
For your previous sequences, showing the same problem, we have already
tested different conditions as:
.- using less material and longer sequencing reactions
.- increasing the annealing and extension temperature in
order to break off the hairpin
.- using additives in order to try to dissolve secondary
structures
All these conditions did not improve the results.
The density of the DNA during the sequencing reactions could influence in
the easiness of breaking off secondary structures. And in same cases, maybe
for A1さん, using less material the reading length can be improved.
For your last sequencing reactions we have used 2ul of the 50ng/uL template
solution under standard conditions. We will repeat the XXX-1 and XXX-2
reactions using 1ul and longer sequencing reaction. |
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