Sanger sequencing is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. It is the most widely used method for the detection of SNVs.
Sanger sequencing is a “first-generation” DNA sequencing method. The method was developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the name the Sanger Sequence.
Sanger’s method is also known as dideoxy sequencing or chain termination. It is based on the use of dideoxynucleotides (ddNTP’s) in addition to the normal nucleotides (NTP’s) found in DNA. Dideoxynucleotides are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a sequence, prevent the addition of further nucleotides. This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.
Major steps in Sanger Sequencing
Complementary strand synthesis
Labeling for the detection of fragments
Chain termination using ddNTPs
Resolution on denaturing PAGE
Visualization of bands by autoradiography
The DNA sequence of interest is used as a template for a special type of PCR called chain-termination PCR. Chain-termination PCR works just like standard PCR, but with one major difference that is the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs). In the extension step of standard PCR, DNA polymerase adds dNTPs to a growing DNA strand by catalyzing the formation of a phosphodiester bond between the free 3’-OH group of the last nucleotide and the 5’-phosphate of the next.
In chain-termination PCR, the user mixes a low ratio of chain-terminating ddNTPs in with the normal dNTPs in the PCR reaction. ddNTPs lack the 3'-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension stops. The result of chain-termination PCR is millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at a random lengths (n) by 5’-ddNTPs.
In manual Sanger sequencing, four PCR reactions are set up, each with only a single type of ddNTP (ddATP, ddTTP, ddGTP, and ddCTP) mixed in.
The chain-terminated oligonucleotides are separated by size through gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size. The smaller a fragment is, the less friction it will experience as it moves through the gel, and the faster it will move. In result, the oligonucleotides will be arranged from smallest to largest, reading the gel from bottom to top.
In manual Sanger sequencing, the oligonucleotides from each of the four PCR reactions are run in four separate lanes of a gel. This allows the user to know which oligonucleotides correspond to each ddNTP.
The last step simply involves reading the gel to determine the sequence of the input DNA. Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence. Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.
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.