In the early days of molecular biology, sequencing worked by cutting the target molecule between specific nucleotides (A and C, for instance). Scientists had isolated enzymes to cut RNAs with this level of specificity – Robert Holley used this strategy to sequence the first RNA molecule in 1965 – but not to cleave DNAs. As a result, DNA sequencing lagged behind RNA sequencing. Until Ray Wu and Dale Kaiser and their revolutionary idea.

Wu and Kaiser flipped the script of sequencing on its head: instead of reading DNAs by breaking them in shorter pieces, DNA sequencing could work by making them longer. How? Using the DNA polymerase [1].

This enzyme, the centrepiece of DNA replication in cells, adds nucleotides to the shorter strand (primer) of a DNA duplex using the longer as a guide (template). By tracking which nucleotides are incorporated and in which order, Wu and Kaiser reasoned, the sequence of the complementary could be revealed.

Using DNA polymerase, Wu and his colleague Ellen Taylor achieved a monumental first in 1971: they deciphered the 12 base pairs at the ends of the lambda bacteriophage. Although this method was rudimentary, imprecise, time-consuming and of limited application, the seed of a revolution – the addition of the DNA polymerase to the sequencing process – had been planted. 1975 bore the first fruits. 

In 1975, Frederick Sanger and his assistant Alan Coulson published a new DNA sequencing process: the “plus/minus” sequencing method. In this method, the DNA polymerase generated a set of DNA chains complementary to a target sequence (the DNA molecule to decipher). These chains stretched all possible lengths, and all ended at or just before a known base. Arranging the chains by size, scientists could determine the sequence of bases in the target DNA. Sanger and Coulson’s process made it possible to sequence stretches of DNA several hundred bases long, a significant step toward reading complete genes.

Here below, you can find an overview of the method, courtesy of its inventors [3]. If you are lost, keep reading: I’ll dissect its intricacies for you.

Figure 1:  The workflow of the plus/minus DNA sequencing method in a schematic representation by Sanger and Coulson (from [3])

Still confused? Here we go!

In more detail, the “plus/minus” sequencing method consisted of two steps, both requiring the DNA polymerase:

1. First step: a primer would be annealed to a template (the DNA to sequence) and then the DNA polymerase would elongate the primer by a varying number of bases. using radiolabeled nucleotides The process produced a set of DNA chains of various lengths, from a few nucleotides to over a hundred nucleotides longer than the original primer.
   
2. Second step: it leveraged the dual nature of the DNA polymerase, which elongates a DNA chain when nucleotides are available and degrades it when they are missing (exonuclease activity). This step was split in 8 parallel reactions, occuring in different tubes: 4 reactions extended the DNA chains from the first step, and 4 shortened them. 
   

• The minus: the DNA fragments and template were incubated with three of the four nucleotides. For instance, in the -C tube, only A, T, and G are available. The DNA polymerase extended each fragment up to the first nucleotide missing from the reaction. As a result, in the -C tube all DNA chains would end up just before a C.
  
• The plus: the DNA fragments and template were incubated with a single nucleotide. For instance, in the +C tube, only C is available. As the nucleotides are not available, elongation of the chains is not possible. Instead, the DNA polymerase removed bases until it reached a position immediately preceding the only available base (C). The remaining fragments thus marked the location of that specific base.

Upon completion of the 8 reactions, scientists loaded the contents of each tube into separate lanes of a polyacrylamide gel and ran an electrophoresis. The use of polyacrylamide was critical because its fine matrix allowed it to separate DNA molecules differing by a single base pair. When visualising the radioactive gel (remember, the nucleotides in step 1 were radio-labelled) in a radioautography, scientists were presented with a (very messy, see Figure 2) ladder of bands over the 8 lanes, in one nucleotide increment. Combining the information on the order of the bands (shorter fragments travel the farther) and on the terminal base (depending on the tube of origin), scientists could finally determine the exact order of As, Ts, Cs and Cs in the target DNA strand. 

Figure 2:  The final output, a ladder of bands spread over 8 lanes. Piecing the correct sequence together was akin to detective work (from [3])

You must have already figured it out, but I’ll spell it out: this method was laborious and time-consuming. It also had two major technical drawbacks:

1. it couldn’t solve homopolymers (which are repeats of the same nucleotide, like AAAAA) and
2. it was sometimes unreliable, because some DNA chains didn’t migrate where expected (shorter fragments would travel farther, leading to underestimating their length, and vice versa). 
   

And yet, it worked! In fact, Sanger, Coulson and colleagues used it to sequence the first DNA genome, the one of the PhiX174 bacteriophage (over 5,300 base pairs), just two years later [4].

Most importantly, the ‘plus/minus” method served as a stepping stone for the development of the far more accurate and rapid dideoxy sequencing method. This method, often simply called Sanger sequencing in honour of its inventor, became the golden standard for DNA sequencing for over 30 years.

In the next episode, we move to 1985 to witness the invention of PCR—the spark that unlocked DNA sequencing on an unimaginable scale.

Want to learn more? Please, subscribe to my blog so you won’t miss the next instalment of this series!

1. Wu & Kaiser (1968). Structure and base sequence in the cohesive ends of bacteriophage lambda DNA. J Mol Biol. Aug 14;35(3):523-37. 
2. Wu & Taylor (1971). Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA. J Mol Biol. May 14;57(3):491-511. 
3. Sanger & Coulson (1975). A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. May 25;94(3):441-8. 
4. Sanger et al (1977). Nucleotide sequence of bacteriophage phi X174 DNA. Nature. Feb 24;265(5596):687-95.