DNA sequencing is a collection of scientific methods for determining the sequence of the nucleotide bases in a molecule of DNA. All living organisms have DNA (deoxyribonucleic acid) in each of their cells. Each cell in an organism contains the genetic code for the entire organism. The process of DNA sequencing transforms the DNA from a given organism into a format that can be used by researchers for the basic study of biologic processes, medical research, and in forensics.
There are several methods that can be used in DNA sequencing. The first methods were developed in the 1970s, and are very laborious and time consuming. The most popular and common sequencing reaction used today is dideoxynucleotide sequencing, which can be done by hand or by machines, depending on the quantity of material to be sequenced.
The amount of genetic material in an organism varies considerably and is measured by the number of nucleotide bases it contains. For example, a virus or bacteria might have as few as five thousand bases, while the human genome contains about three billion bases. The dideoxynucleotide sequencing method of DNA sequencing can sequence many genomes in days, and large genomes in years, rather than decades.
There are four stages in DNA sequencing. First the DNA must be removed from the cell. Then it undergoes a sequencing reaction. Next, the DNA is separated by size, and finally analyzed by a computer that puts the results into a usable format.
The first step in DNA sequencing is to get the DNA out of the cell. This can be done mechanically or chemically. DNA comes in two strands, but only one strand can be sequenced at a time.
Once the DNA is broken up, it is put on vectors, which are cells that will self-replicate indefinitely, along with a primer, which is a chemical that gets the process started. This creates clones of the DNA of the organism that is being sequenced. The sequencing reaction uses the primer to start the chemical process of reproducing the second DNA strand. The sequencing is performed in a thermal cycler so that the reaction is repeated many times. Repeating the reaction results in a greater yield of sequenced DNA.
After sequencing, the DNA is sorted by size by capillary electrophoresis. The DNA is pulled by an electric current through a gel in the capillary, which is a very thin glass tube. The DNA strands emerge sorted by length. As they emerge from the capillary, they pass through a laser that activates dyes that identify the nucleotide bases. This information is fed into a computer, which then displays the DNA sequence on screen.