DNA sequencing involves determining the linear nucleotide order of a segment of DNA. There are several methods of sequencing, but most are based on the Sanger Method. This is an enzymatic method that synthesizes DNA in vitro. The synthesized DNA is complementary to the template DNA. By determining the nucleotide sequence of the synthesized DNA, we can deduce the sequence of the template DNA.

Reaction Components
Template is single-stranded DNA that you want to sequence. It can be a PCR product, genomic DNA, or cloned fragment.

Primer is a short fragment of DNA that binds to one end of the template DNA. The primer provides specificity to the sequence reaction and also serves as the anchor to which nucleotides are added.

Deoxynucleotides (dNTPs) extend the primer, forming a DNA chain. All four nucleotides (A,T,G,C in deoxynucleotide form) are added to the sequencing reaction.

Dideoxynucleotides (ddNTPs) are another form of nucleotide that inhibit extension of the primer. Once a ddNTP has been incorporated into then DNA chain, no further nucleotides can be added.

DNA polymerase incorporates the nucleotides and dideoxynucleotides into the growing DNA chain.

Buffer is a solution that stabilizes the reagents and products in the sequencing reaction.

Procedure
           The components of the reaction are combined and allowed to incubate. Many copies of the template DNA are made by primer extension (adding nucleotides on to the primer). The copies all have the same nucleotide sequence but vary in length because the ddNTPs incorporate randomly and stop extension. Products of the reactions are then run on a gel that separates the DNA fragments by size.


The sequencing products of the dideoxynucleotide ddTTP are shown. Notice that each product ends with a T (thymine) which corresponds to an A (adenine) in the template sequence.

Manual Sequencing
           In manual sequencing, the reaction takes place in four different tubes, each of which contains a different ddNTP. Thus the tube that contains ddATP will have fragments that all terminate at an adenosine (A), the tube with ddGTP will have fragments that end with a guanine (G) and so on. The products from the four tubes are run in parallel lanes of a gel. The figure on the right shows the results of manual sequencing.
           The products of DNA sequencing can be visualized because the primer is tagged with a radioactive label. When exposed to a piece of X-ray film, the radioactivity exposes the film showing up as a dark band. The sequence is then read from the film by the researcher or technician.


Photo of a manually sequenced DNA. A portion of the sequence is written down the right side.
How to read a manual sequence
           The products of the sequence are loaded in four parallel lanes on a gel. In this example, the first lane contains the products from reaction containing ddCTP. Thus every band that shows up in that lane represents a sequencing product that terminates at a C. The gel separates the sequencing products based on size; smaller fragments travel through the gel faster then longer fragments. The four lanes are read together in a horizontal heirarchy from bottom (smallest) to top (largest). The blue line traces the order that the bands are read and the sequence of the fragment is shown on side.

Automated sequencing
           The reaction in automated sequencing is essentially the same as in manual sequencing. There are two main differences: the labeling and reading. In automated sequencing, the products are labeled with a fluorescent dye rather then a radioactive label. There are four fluorescent dyes, each corresponding to a different ddNTP; ddATP is green, ddTTP is red, ddCTP is blue, and ddGTP is yellow. Thus each fragment has a different color at its end depending on which is the terminating nucleotide (ddNTP). This allows the products of sequencing to be run on a single lane of a gel rather then in four parallel lanes. In addition, the sequencing of nucleotides is determined by the computer, rather then being read manually by a technician. As the samples pass through the gel, a laser excites the fluorescent labels. A computer collects and analyzes this data, reading the sequence of the DNA. Thus automated sequencing is much faster and more efficient then manual sequencing. The human genome is being sequenced using automated sequencing.
This image, called an electropherogram, is the computer generated output of automated sequencing. The peaks represent the intensity of the fluorescent ddNTPs. The sequence is printed across the top of the peaks.
This figure illustrates why automated sequenced products can be run in a single lane of a gel, while manually sequenced products must be run in four adjacent lanes of a gel.

           So what do different types of mutations look like? See what different types of mutations look like when sequenced manually or by automated techniques in the figures below. Remember, we typically have two copies of all autosomal genes. When we sequence a gene, both copies of the gene are analyzed simultaneously. When the two alleles are identical, they overlap perfectly. However, when one allele is different from the other they no longer overlap perfectly and the differences can be identified.

Point Mutation

Manual Sequencing

    The arrow head indicates the location of the point mutation. The wild-type sequence is a G at this location, but the mutated sequence has both a G and an A. This sample is heterozygous at this locus; there is one wild-type copy of the gene (G) and one mutant copy of the gene (A).

Automated Sequencing

    The point mutation is shown by the arrowhead and is indicated as an N in the printed sequence. In the wild-type sequence the base is a T, but in the mutated sequence we see two overlapping peaks of flourescence, representing both a T and a C. This sample is heterozygous at this locus (one mutant and one wild-type allele).

Insertion
Automated Sequencing

    The wild-type sequence is illustrated on top and the bottom is the same sequence but with a 1 basepair insertion of a G at the arrow. The insertion only occurs on one allele, so the sample is heterozygous at this locus. Notice that the insertion throws off the alignment of the two allele sequences. The sequence prior to the insertion is in alignment, but after the insertion they are out of alignment and you see two overlapping sequences.

Deletion
The bracket on the wild-type sequence shows the 2 bases (A and G) that are deleted in the mutant sequence. As with insertions, heterozygous deletions results in overlapping of the 2 allele sequences which looks like stuttering or staggering in manual sequencing, as shown in this picture.