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Sequence confirmation of synthetic phosphorothioate oligodeoxynucleotides using Sanger sequencing reactions in combination with mass spectrometry
- Source :
-
Analytical Biochemistry . Oct2005, Vol. 345 Issue 1, p81-89. 9p. - Publication Year :
- 2005
-
Abstract
- Abstract: A protocol relying on Sanger sequencing reactions in combination with mass spectrometry (MS) for sequence confirmation of antisense phosphorothioate oligodeoxynucleotides is described. In this procedure, synthetic phosphorothioate oligodeoxynucleotides are used as reverse primers for extension of matched templates with enough length (∼150–300bp) for well-established Sanger sequencing. Because the complementary strand of modified primer is used directly for sequencing primer extension, the base order shown in the sequencing result is reversely complementary to phosphorothioate oligodeoxynucleotide. This sequencing method can be applied not only to phosphorothioate oligodeoxynucleotides with different lengths (13–21mer) and base composition but also to sequences with bases’ switch, deletion, or insertion. In addition, modified primers incorporate the 5′ end of polymerase chain reaction (PCR) products conveying the characters of phosphorothioate modification. The method requires only common reagents and instruments and so is better suited to routine sequence analysis in quality control of phosphorothioate antisense drugs. [Copyright &y& Elsevier]
- Subjects :
- *MASS spectrometry
*NUCLEOTIDES
*NUCLEIC acids
*POLYMERASE chain reaction
Subjects
Details
- Language :
- English
- ISSN :
- 00032697
- Volume :
- 345
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Analytical Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 18345521
- Full Text :
- https://doi.org/10.1016/j.ab.2005.06.045