I used an oligonucleotide in a cloning experiment an when I sequence through the area represented by the oligo the sequence is different from the sequence I ordered. Why?
There are number of possible explanations for apparent errors in the sequence of the oligonucleotide used in cloning experiments. There could have been human error during the ordering and synthesis of the oligonucleotides. Human error can be easily checked by looking at the sequence on the specification sheet that accompanies each oligo. Below is a list of problems associated with using the beta-cyanoethyl phosphoramidite chemistry that gives the impression that a synthesis error had been introduced. 13.1 The G base may have been converted to the enol tautomer, 2,6 diaminopurine, which is recognized as A by DNA polymerase. Thus, clones generated from an oligo with this modified base will appear as a G to A transition. 13.2 The chemical process of synthesis may cause depurination (A and G, purine). Depurinated oligos are usually degraded at the deprotection stage, but it is possible for a small percentage to remain. Clones containing an inserted oligo that was depurinated will appear as
Related Questions
- Why is the subtype B consensus sequence used rather than a particular isolate, the consensus sequence for a different subtype, or the consensus sequence for Group M sequences?
- I used an oligonucleotide in a cloning experiment an when I sequence through the area represented by the oligo the sequence is different from the sequence I ordered. Why?
- I used oligos for cloning, sequenced a clone and found a mutation within the oligo sequence. Can mutations happen in synthetic oligos?
