When a fragment of gene was cloned into a vector for sequencing, two scenarios may lead to a change of the sequence. Errors in the PCR amplification and wrong fragments can be cloned into vectors. ABI promises that the sequencing accuracy of its instrument can reach over 98.5% within a certain range. Because of the limitation of the accuracy of the instrument, it is difficult to avoid a base sequence error in a longer sequence. After confirming the accuracy of cloning, bidirectional sequencing can minimize the sequencing errors. If you want to get your most accurate sequence, it is necessary to carry out bidirectional sequencing. With simple unidirectional sequencing, we can not guarantee the complete accuracy of the measured sequence, which is determined by the accuracy of the instrument.
Articles in this section
- Will the primers degrade at room temperature during transportation? Can we use dry ice for transporting to maintain stability?
- Why there is a difference between my sequencing data and the one from the references?
- Why the short PCR product is not suitable for direct sequencing?
- Why target gene products are seen in control sample with no template?
- Why my sequence results are reverse complement to the template DNA, while I demand a 5’ to 3’ orientation?
- Why is the TM value for primers of Genscript different from the TM value I obtained from other software?
- Why is the sequence of primers in the sequencing results different from the expected sequence?
- Why is my gene sequence different from the standard sequence?
- Why I got a sequencing result that was not I want?
- Why does the sequencing primer need to be site-specific on the DNA template?