MAKING SENSE OF SANGER SEQUENCING

 WHAT IS SANGER SEQUENCING?

Sanger sequencing is a method that yields information about the identity and order of the four nucleotide bases in a segment of DNA. Also known also as the “chain-termination method”, it was developed in 1977 by Frederick Sanger and colleagues, and is still considered the gold standard of sequencing technology today since it provides a high degree of accuracy, long-read capabilities, and the flexibility to support a diverse range of applications in many research areas

In the mid-1970s, Sanger wasn’t alone in the race to sequence DNA; almost in parallel, two American scientists, Maxam and Gilbert, developed a technique in which DNA is chemically treated to break the chain at specific bases. Following electrophoresis of the cleaved DNA, the relative lengths of the fragments—and thus the positions of specific nucleotides—can be determined and the sequence inferred. This is considered the birth of first-generation sequencing. However, the advent of Sanger’s chain-termination method in 1977 would be the breakthrough that propelled sequencing into the future; many years after its development, Sanger sequencing was used to sequence the entire human genome.

OVERVIEW OF SANGER SEQUENCING

Sanger sequencing targets a specific region of template DNA using an oligonucleotide sequencing primer, which binds to the DNA adjacent to the region of interest. (There must be an area of known sequence close to the target DNA.)W In order to determine the sequence, Sanger sequencing makes use of chemical analogs of the four nucleotides in DNA. These analogs, called deoxyribonucleotides (ddNTPs), are missing the 3´ hydroxyl group that is required for 5’ to 3’ extension of a DNA polynucleotide chain. By mixing ddNTPs that have been labeled with a different color for each base, unlabeled dNTPs, and template DNA in a polymerase-driven reaction, strands of each possible length are produced when the ddNTPs are randomly incorporated and terminate the chain. The extension products are then separated by electrophoresis, and resolved to single-nucleotide differences in size. The chain-terminated fragments are detected by their fluorescent labels, with each color identifying one of the terminating ddNTPs. The sequence of the template DNA strand can thus be derived by analysis.

WORKFLOW OF SANGER SEQUENCING

- cycle sequencing

If they synthesize DNA in their sequencing reaction by doing “cycle” sequencing using Taq polymerase, we can get a much larger amount of product. There are many more pieces made of each product strand and so these show up better on the gel.  When researchers first began to do this they were still dealing with the health and safety concerns of working with isotopes. This meant we had to shield the thermocycler and do careful swipe testing after using it. Swipe testing was a way to monitor whether equipment or working spaces were contaminated with radioactivity. If they were contaminated that was a serious issue and an arduous clean up/decontamination process took place.

- fluorescent terminators

To eliminate the safety concerns of working with isotopes researchers turned to fluorescence. They used differently labeled ddNTPs for each reaction.  The ddATP would be labeled with red fluorescence, while the ddCTP might be labeled with yellow fluorescence. So each strand of DNA that was synthesized in the sequencing reaction would end with a ddNTP and a unique, nucleotide-specific fluorescent label. This meant that the reaction could all be done in a single tube. So not only is the work safer but it is more efficient as well.

To detect the fluorescence a computer apparatus is set up. Each reaction is run in a polyacrylamide gel in a single lane and towards the end of the gel, there is a laser that excites the fluorescence and a photomultiplier that detects the light emitted and sends the information to the computer for analysis. Each molecule is “read” as it passes the detector and the color is recorded. This computer produces a trace, showing the fluorescence detected in peaks, where each peak corresponds to a nucleotide.

KEY DIFFERENCES BETWEEN SANGER SEQUENCING AND NEXT-GENERATION SEQUENCING

In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar. In both NGS and Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing), DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Each incorporated nucleotide is identified by its fluorescent tag.

The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This process translates into sequencing hundreds to thousands of genes at one time. NGS also offers greater discovery power to detect novel or rare variants with deep sequencing.

COMPARISON 

        SANGER SEQUENCING BENEFITS             NGS BENEFITS

s. no
1 2	Fast, cost-effective sequencing for low numbers of targets (1–20 targets) Familiar workflow	Higher sequencing depth enables higher sensitivity (down to 1%) Higher discovery power* Higher mutation resolution† More data produced with the same amount of input DNA‡ Higher sample throughput

CONCLUSION

Sanger sequencing has undergone many changes over the last 40 years, but it remains the most commonly used DNA sequencing technology worldwide. With 99.99% accuracy, it is the gold standard for most applications—both research and clinical. However, Sanger sequencing is best suited for medium- to low-throughput targeted sequencing projects; higher-throughput DNA sequencing technologies based on fundamentally different methods have emerged in the last decade. Called next-generation sequencing (NGS), these massively parallel technologies have revolutionized the study of genomics and molecular biology.