Sanger Sequencing Steps & Method

What is Sanger Sequencing?

Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA. The method was developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the name the Sanger Sequence.

Sanger Sequencing Steps

There are three main steps to Sanger sequencing.

1. The first step is generating n DNA fragments of varying lengths, each terminated with a labeled nucleotide, where n is the number of nucleotide bases in the target DNA sequence. This is done by combining DNA primer, nucleotides (dATP, dCTP, dGTP, dTTP), DNA polymerase, the DNA sequence of interest, and labeled dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP). No nucleotide can be added to the DNA chain once a dideoxynucleotides has been incorporated, so each fragment will end with a labeled nucleotide. A much smaller amount of dideoxynucleotides is used than the amount of regular nucleotides.

2. The second step is to separate the n DNA sequences be length using capillary gel electrophoresis. The shorter fragments move faster than the longer fragments. The result is that the DNA pieces are fed into the third step from shortest to longest sequence.

3. In the third step a laser excites the label on the nucleotide at the end of each sequence. Each base is tagged with a different label, so the light emitted by each excited nucleotide can be tied to the correct base. The laser generates a chromatogram showing the fluorescent peak of each nucleotide. The chromatogram has the nucleotides in the correct order because of the electrophoresis.

(Note that the reaction can be done by putting all 4 dideoxynucleotides in one mix with the template, or 4 separate reactions can be set up. If four reactions are set up, then each group of dideoxynucleotide fragments is put in a different lane in the gel for separation and reading. The principles remain the same though.)

DNA structure with base, sugar and phospate enlarged. In sanger Sequencing "n" fragments of DNA are generated each with a terminating with a labeled nucleotide.

DNA structure with base, sugar and phospate enlarged. In sanger Sequencing "n" fragments of DNA are generated each with a terminating with a labeled nucleotide.

How Does Sanger Sequencing Work?

Sanger sequencing works on the principle that when given enough time and enough starting material, at least one DNA sequence of every possible length will be produced with a tagged nucleotide at the end. The tagged nucleotide will always terminate the sequence because dideoxynucleotides are missing the 3'-OH group that is necessary to continue the chain.

How to Read Sanger Sequencing Results

Reading the Sanger sequencing results properly will depend on which of the two complementary DNA strands is of interest and what primer is available. If the two strands of DNA are A and B and strand A is of interest, but the primer is better for strand B, the output fragments will be identical to strand A. On the other hand, if strand A is of interest and the primer is better for strand A, then the output will be identical to strand B. Accordingly, the output must be converted back to strand A.

So, if the sequence of interest reads “TACG” and the primer is best for that strand, the output will be “ATGC” and, therefore, must be converted back to “TACG”. However, if the primer is better for the complementary strand (“ATGC”), then the output will be “TACG”, which is the correct sequence.

In short, before starting, you need to know what you’re targeting and how you’re going to get there! So keeping this in mind, here is an example of the former example (TACG -> ATGC -> TACG). If the dideoxynucleotides labels are T = yellow, A = pink, C = dark blue, and G = light blue, you will end up with the short sequences primer-A, primer-AT, primer-ATG, and primer-ATGC. Once the fragments have been separated by electrophoresis, the laser will read the fragments in order of length (pink, yellow, light blue, and dark blue) and produce a chromatogram. The computer will convert the letters, so the final sequence is the correct TACG.

Sanger Sequencing vs. PCR

Sanger sequencing and PCR use similar starting materials and can be used in conjunction with each other, but neither can replace the other. PCR is used to amplify DNA in its entirety. While fragments of varying lengths may be produced by accident (e.g., the DNA polymerase might fall off), the goal is to duplicate the entire DNA sequence. To that end, the “ingredients” are the target DNA, nucleotides, DNA primer, and DNA polymerase (specifically Taq polymerase, which can survive the high temperatures required in PCR). In contrast, the goal of Sanger sequencing is to generate every possible length of DNA up to the full length of the target DNA. That is why, in addition to the PCR starting materials, the dideoxynucleotides are necessary. Sanger sequencing and PCR can be brought together when generating the starting material for a Sanger sequencing protocol. PCR can be used to create many copies of the DNA that is to be sequenced. Having more than one template to work from makes the Sanger protocol more efficient. If the target sequence is 1,000 nucleotides long and there is only one copy of the template, it is going to take longer to generate the 1,000 tagged fragments. However, if there are several copies of the template, in theory it will take less time to generate all 1,000 of the tagged fragments.

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