DNA Sequencing 101: An Examination of the Sanger Method

in Technology

The Sanger method of DNA sequencing is also known as Sanger sequencing or the chain termination method. This method is named after its founder: Frederick Sanger. Sanger won a Nobel prize in 1980 for discovering the chain termination method.

Sanger sequencing allows scientists and researchers to determine the sequence of nucleotides in a strand of DNA. How exactly do they do it? The Sanger method has three main steps, which we’re talking about next.

  1. Chain Termination PCR

Polymerase chain reaction (PCR) is a technique used to create copies of a certain DNA sequence. This allows researchers to use them for gel electrophoresis.

The Sanger method utilizes a unique type of PCR called chain-termination PCR. It’s nearly identical to regular PCR, with the addition of special modified nucleotides called ddNTPs. A ddNTPs could be:

  • ddATP (modified adenine)
  • ddTTP (modified thymine)
  • ddGTP (modified guanine)
  • ddCTP (modified cytosine)

What’s the purpose of chain-termination PCR? ddNTPs act as a stop sign for DNA chain reactions. Importantly, ddNTPs stop each chain at random, meaning the end-result is multiple strands of DNA of differing lengths.

  1. Gel Electrophoresis

Gel electrophoresis separates each of the chain-terminated DNA sequences by size. Researchers place all the DNA sequences from step 1 into a gel matrix. When the researchers apply an electric current to the gel, the sequences move to the opposite side of the matrix.

Heavy objects move slower than lighter objects. The same applies here—the lighter, smaller DNA sequences travel faster than the heavier, larger sequences. This allows the researchers to identify each DNA sequence from smallest to largest.

  1. Analysis and Determination

The final step of the Sanger method is to determine the sequence of the original target DNA sequence. This requires analyzing the relative positions of the DNA strands from step 2.

Specifically, researchers look for the ddNTP in each strand. Why? Because it specifies one of the nucleotides in the target DNA sequence.

Sometimes, the exact nucleotide sequence is indecipherable using the Sanger method alone. In this case, researchers and scientists can take advantage of alternative sequencing methods like polyphasic analysis.

Automated vs Manual Sanger DNA Sequencing

Sanger sequencing can be either manual or automated.

Manual Sanger sequencing requires four separate PCR reactions with only a single one of the four ddNTPs. During electrophoresis, manual Sanger sequencing requires four separate lanes to differentiate the four different ddNTPs.

Researchers more commonly turn to automated Sanger sequencing, which uses a sequencing machine. Another difference is that each ddNTP gets an added fluorescent label to differentiate it from the others. This allows scientists to use only one lane for gel electrophoresis.

The Sanger Method Is the Gold Standard of DNA Sequencing

The Sanger method of discovering the sequence of a certain DNA strand is the gold standard in research. Combined with polyphasic analysis and other sequencing methods, researchers can learn the identity of an unknown DNA strand.

Image Credits: PublicDomainPictures

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