DNA sequencing is a process of determining the sequence of nucleotide bases in a DNA molecule. There are several methods, with dideoxynucleotide sequencing being the most popular. The process involves extracting DNA from cells, sequencing it, sorting it by size, and analyzing it with a computer.
DNA sequencing is a collection of scientific methods for determining the sequence of nucleotide bases in a DNA molecule. All living organisms have DNA (deoxyribonucleic acid) in each of their cells. Each cell of an organism contains the genetic code of the entire organism. The DNA sequencing process transforms the DNA of a given organism into a format that can be used by researchers for the study of basic biological 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 labor intensive and time consuming. The most popular and common sequencing reaction used today is dideoxynucleotide sequencing, which can be done manually or by machines, depending on the amount 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 bacterium might have only five thousand bases, while the human genome contains around 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 has to 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 extract the DNA from the cell. This can be done mechanically or chemically. DNA is made up of two strands, but only one strand can be sequenced at a time.
Once the DNA is broken down, it is put onto vectors, which are cells that self-replicate indefinitely, along with a primer, which is a chemical that starts the process. This creates clones of the organism’s DNA which is sequenced. The sequencing reaction uses the primer to start the chemical process of making the second strand of DNA. Sequencing is performed in a thermal cycler so that the reaction is repeated many times. Repeating the reaction results in a higher 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. DNA strands emerge ordered by length. As they emerge from the capillary, they pass through a laser that activates the dyes that identify the nucleotide bases. This information is entered into a computer, which then displays the DNA sequence on screen.
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