Sanger Sequencing Background
Sanger Sequencing, also known as “first-generation”, “conventional” or “cycle” sequencing, uses a mixture of natural nucleotides and fluorescently-labeled, dideoxynucleotides to generate DNA sequence. These di-deoxynucleotides (ddNTPs) do not have a 3′-hydroxl (-OH) group on the DNA’s sugar backbone; thus, elongation of the template DNA via the DNA polymerase does not occur after a di-deoxynucleotide is incorporated. When PCR product—amplified from genomic DNA or vectors—is generated in the presence of natural dNTP’s and four fluorescently-labeled ddNTPs, fragments of random length are generated. These fragments are then separated by size using electrophoresis and detected by a laser, with each fragment labeled with the specific ddNTP that prevented strand elongation. More background information regarding the principle of Sanger Sequencing can be found here.
Sanger Sequencing can be applied to your research to:
- Determine sequence content of DNA inserts (or success) during cloning experiments
- Determine PCR amplicon sequence content
- Annotate DNA regions for sites of variation such as single nucleotide polymorphisms (SNPs), insertions/deletions of several bases, and short tandem repeats (STRs)
- Sequence or re-sequence regions of interest or gaps based on analyses of Roche 454 or other “Next-Gen” sequencing data
If SNP sites have allele frequencies of <10%, or contain multiple repeated elements (e.g., poly-nucleotides), Sanger Sequencing will not be able to sequence these rare variants effectively. However, other services offered at the Laboratory of Molecular Technology, such as Pyrosequencing or Roche 454 Sequencing, maybe more suitable for rare variant detection and allele quantification, or for thorough, deep-sequencing of regions.
For more information on this service and its applications to your research, visit our Sanger Sequencing Protocols and Resources page.