• The following slides illustrate the process of amplifying a nested set of DNA fragments for electrophoresis on a sequencing gel. Photographs from the biology division sequencing facility show the process of preparing a sequencing gel. This presentation was developed as part of an institutional technology fellowship to Karen Hughes and may be used for educational purposes without permission.
• The Sanger method of DNA sequencing involves copying a fragment of DNA over and over, each time terminating the copied sequence prematurely by incorporating a dideoxynucleoside triphosphate (above) instead of a normal deoxynucleoside triphosphate.
• Dideoxynucleoside triphosphates lack an -oh group at the 3-carbon position and cannot add another nucleoside at that position, thus preventing further DNA synthesis.
• This random premature termination creates a set of nested fragments of DNA, each differing by a single base in length. These fragments can be separated on a polyacrylamide gel.
• The next slides show the process of creating a set of nested fragments.
• The DNA polymerase enzyme is used to copy a fragment of DNA.
• DNA polymerase requires a primer to attach to before it can copy DNA.
• The first step in the copying process is the pairing of a primer with the homologous sequence on a segment of DNA.
• DNA polymerase attaches to the primer and begins copying the DNA strand.
• DNA polymerase uses a mixture of nucleoside triphosphates to synthesize a new DNA strand.
• The DNA copy is extended.
• Dideoxynucleotides are mixed at a low concentration with normal nucleoside triphosphates.
• Occasionally one is incorporated in place of a normal nucleotide triphosphate.
• When that happens, the new DNA copy is terminated.
• The DNA copy is released and the original strand is available to be copied again.
• The DNA segment is copied again until another dideoxy nucleoside is incorporated.
• The second DNA copy is released.
• The DNA strand is copied again and terminated as another dideoxy base is incorporated.
• The third strand is released. This process is repeated until thousands of copies have been made, each terminated at a different base.
• The three DNA copies previously diagrammed are shown above. They represent only a fraction of the possible DNA fragments.
• Each fragment will end in a specific dideoxy nucleotide. That nucleotide can be recognized by the fluorescent dye attached to it.
• When the three fragments (previous slide) are electrophoresed on a gel, they will separate by size. The smallest fragments will be at the bottom of the gel, the largest fragments at the top.
• The DNA sequence can be determined by determining the terminating base for the shortest fragment, then for the next shortest fragment for all of the DNA fragments, not just the three diagrammed above.
• The next slides depict the actual preparation of a sequencing gel, the loading of the sequencing gel with the DNA fragments, and the computerized output from the gel.
• A polyacrylamide gel is a very thin gel poured between two plates of glass. It is a large gel which will separate DNAs that differ by a single base in length. This photo shows one of the glass plates that will be used for the gel. The white strips on the lower plate are spacers which separate the two glass plates.
• The top plate is lowered onto the bottom plate.
• The two plates are taped together to prevent leakage.
• Polyacrylamide is poured between the two plates. This will form a matrix with small pores. DNA fragments will move through this matrix when an electrical field is applied. Large fragments will lag proportionately behind small fragments.
• When the gel has hardened, the tape is removed and the plates are placed in the abi automated sequencing machine. Buffer trays at the top and bottom are installed so that electrical current can run through the gel.
• DNA samples (nested fragments of DNA copied from a single sequence) are prepared for loading.
• The DNA samples are loaded on the gel. As these samples are electrophoresed, they will migrate down the gel where they will pass through a laser beam. The laser beam will record the fluorescent dye attached to each dideoxy nucleotide at the end of a sequence and therefore, which base is in that position.
• The fluorescent bands in each lane of the DNA are read from the bottom up to determine the sequence of the DNA segment.
• A printout of the laser scan is also prepared as a permanent record.

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