Sanger sequencing
Read a miniprep with vector primers on both sides of the insert, plus internal primers for long inserts, until every base and both junctions are read at good quality, then align to the designed map.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Plasmid verification
Read a miniprep with vector primers on both sides of the insert, plus internal primers for long inserts, until every base and both junctions are read at good quality, then align to the designed map.
How it differs from the standard method
- Template is a supercoiled plasmid: typically 100–300 ng for a 5 kb vector in an in-house reaction (core facilities quote their own amounts).
- Vector primers sit 50 bp or more upstream of the junction so the blind start falls on the vector, not the insert.
- A clone is verified only where it was read; reads from both ends usually overlap in the middle of inserts up to ~1.5 kb.
- Double peaks from the junction onward mean two plasmids in the prep: re-streak the colony.
1Chain termination, read by colour
A single primer anneals to the template and a DNA polymerase extends it, mostly with ordinary dNTPs but occasionally with a dideoxynucleotide that has no 3′ hydroxyl. Each dideoxy base carries one of four fluorescent dyes and stops the chain where it is added.
One read
A good reaction gives about 700–900 bases at QV20 or better. Longer inserts need more than one primer.
The blind start
The shortest fragments are swamped by primer and free dye, so the bases just after the primer cannot be read.
One template
Each position should show a single clean peak. Two peaks mean two templates: a heterozygote, a mixed colony or a second primer site.
2How a read fails
- Too little template: small peaks, a noisy baseline and a read that never reaches high quality.
- Too much template: a huge early signal that fades within a few hundred bases, because the terminators are used up on short products (a 'top-heavy' read).
- Inhibitors in the template: EDTA from TE takes the magnesium, while ethanol, salt and phenol all weaken the reaction.
- Dye blobs: unincorporated terminators left after clean-up run as broad peaks around bases 60–90 and hide what is under them.
- Mixed signal: a primer with two binding sites, two plasmids in one prep, or left-over PCR primers all put a second sequence under the first.
- Hard templates: GC-rich stretches, hairpins and long homopolymers make the signal drop suddenly or the peaks smear.
3Reading a chromatogram
The text file is an interpretation, so check the trace wherever the answer matters. Look at the quality first: a call means nothing where the peaks are low, broad or overlapping.
Key terms
Practise these as flashcards →A nucleotide lacking the 3′ hydroxyl, so the chain cannot be extended past it; dye-labelled in modern chemistry.
The four fluorescence channels recorded as fragments pass the detector; one peak per base.
Phred-scaled confidence in a base call: QV = −10·log10(error probability).
The number of bases in a read called at QV20 or better; the usual summary of read quality.
A broad peak from unincorporated dye terminator left after clean-up, typically near bases 60–90.
Very strong early peaks that fade quickly, the typical result of too much template.
A position with two overlapping peaks; written as an IUPAC code (R = A/G, Y = C/T) by callers that report them.
When to use it
- Confirming that a cloned insert, a site-directed mutant or a gene-edited allele has exactly the designed sequence, junctions included.
- Genotyping one or a few known variants in a PCR product from a handful of samples.
- Checking a primer, a vector or an amplicon before investing in a larger experiment.
- Validating a variant first found by next-generation sequencing.
Compared with alternatives
Millions of reads at once; prefer it for many samples or targets, or to detect variants present in well under ~15–20% of molecules.
Whole-plasmid sequencing with long reads covers an entire construct in one read and is now often cheaper than several Sanger reactions for full plasmids.
PCR- or probe-based genotyping is faster and cheaper per sample when the variant is known and only its presence matters.
Sanger is how a mutagenesis clone is checked: the designed change plus the rest of the open reading frame.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Read a miniprep with vector primers on both sides of the insert, plus internal primers for long inserts, until every base and both junctions are read at good quality, then align to the designed map.
- Template is a supercoiled plasmid: typically 100–300 ng for a 5 kb vector in an in-house reaction (core facilities quote their own amounts).
- Vector primers sit 50 bp or more upstream of the junction so the blind start falls on the vector, not the insert.
- A clone is verified only where it was read; reads from both ends usually overlap in the middle of inserts up to ~1.5 kb.
- Double peaks from the junction onward mean two plasmids in the prep: re-streak the colony.