Common challenges of DNA sequencing with the Sanger method include poor quality in the first 15-40 bases of the sequence due to primer binding and deteriorating quality of sequencing traces after 700-900 bases. Base calling software such as Phred typically provides an estimate of quality to aid in trimming of low-quality regions of sequences.
In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR-based cloning and next-generation sequencing technologies based on pyrosequencing often avoid using cloning vectors. Recently, one-step Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.
Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.
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Famous quotes containing the word challenges:
“A powerful idea communicates some of its strength to him who challenges it.”
—Marcel Proust (18711922)
“The approval of the public is to be avoided like the plague. It is absolutely essential to keep the public from entering if one wishes to avoid confusion. I must add that the public must be kept panting in expectation at the gate by a system of challenges and provocations.”
—André Breton (18961966)