Microfluidic Sanger Sequencing - Method - Challenges

Challenges

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.

Read more about this topic:  Microfluidic Sanger Sequencing, Method

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