Sanger sequencing
Sanger sequencing is a DNA sequencing method which determines the nucleotide sequence in DNA molecules by the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during DNA amplification.
Sanger sequencing is a DNA sequencing method which determines the nucleotide sequence in DNA molecules by the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during DNA amplification.
No. PCR is used to amplify the DNA region of interest prior to Sanger sequencing. Although the reagents used in Sanger sequencing are similar to those needed for PCR, a Sanger sequencing reaction also contains a unique ingredient: the dideoxy (or chain-terminating) versions of all four nucleotides (i.e. ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye. It is the dideoxy nucleotides that result in the different fragment lengths.
The Sanger method relies on using dideoxy chain terminating nucleotides to produce sequence fragments of graduated lengths, each equal to the position of the base complementing the terminating nucleotide. Reading the DNA sequence involves visualizing these fragments and identifying the terminating nucleotides in order of fragment length.
ddNTPs are used in Sanger sequencing as they stop polymerisation of DNA strands during DNA replication. This results in the production of different lengths of DNA strands replicated from a template strand.