The shotgun method breaks up DNA into random fragments and sequences them using the chain termination method. Multiple reads are used to create a continuous sequence, but errors can occur due to repeating sequences. Next-generation sequencing is now faster but less accurate.
The shotgun method is used to sequence long strands of a deoxyribonucleic acid (DNA) molecule. It is based on another DNA sequencing method called the chain termination method. With the shotgun method, scientists sequence parts of the DNA strand and use the results to create a continuous sequence.
Sequencing is the process of finding the atomic composition of a molecule and studying the chemical bonds that join those atoms together. In DNA, sequencing is used to find the order in which certain molecules occur. These molecules are called nucleotides. The four nucleotide bases are adenine, guanine, cytosine and thymine.
One way to sequence long strands of DNA is to use the shotgun method. In this method, DNA is broken up into random fragments. The resulting segments are then sequenced using a method called the chain termination method. Chain termination is the method of choice for short DNA sequences. It uses few harsh chemicals and less radioactivity than other short-sequence methods.
In the chain termination method, a DNA strand is split into four sequencing reactions. Each contains all four nucleotides and an enzyme that acts as a catalyst, called DNA polymerase. A dideoxynucleotide is then added to each sequencing reaction, which is basically a nucleotide devoid of part of its sugar sequence. After processing these reactions, scientists are able to visualize them and read the DNA sequence.
During the shotgun method of DNA sequencing, this all happens multiple times using different random fragments of DNA. This results in multiple overlapping sections of sequenced DNA. A computer program then uses the overlapping sections to create a continuous DNA sequence.
The problem with the shotgun method is that DNA is incredibly complex. It often contains repeating sequences that look the same but come from different parts of the DNA. The complete set of human DNA, called the human genome, has more than three billion base pairs. The only way to be sure that the placement of repeating sequences is correct is to perform multiple random reads so that each part is sequenced multiple times. Even so, errors do occur.
The shotgun sequencing method was at the forefront of DNA sequencing until around 2005. Since then, scientists have created next-generation sequencing, which works much faster. The accuracy of single reads is lower than with shotgun sequencing, but scientists are compensated by being able to take more reads in less time.
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