DNA sequencing is a molecular biology method of defining the specific order and arrangement of nucleotide bases (adenine, guanine, cytosine, and thymine) within a DNA molecule. With DNA sequencing the DNA from an species is transformed into a format suitable to be used by researchers for their basic molecular biology studies, medical research, and in forensics investigations. The advancement of rapid DNA sequencing techniques has significantly accelerated genetics and medical research and discovery. The DNA sequencing technology has evolved rapidly in the last couple of decades and has achieved rapid speed of sequencing. This makes possible the sequencing of complete DNA sequences, or genomes of important model organism, including the human.
What Is DNA Sequencing – The Basics
In general, there are four stages in the process DNA sequencing. The first step is the isolation of the cell DNA. Next follows the actual DNA sequencing reaction. After that, the resulting DNA fragments are separated by their size. In the final step, the DNA profile is analyzed by a computer and the final DNA sequencing data is then presented in a usable format.
The first step in DNA sequencing process is to extract the DNA out of the cell. This can be achieved either mechanically or biochemically. Usually the DNA molecule is a double stranded – it has two chains of nucleotide residues. With the DNA sequencing only one strand is sequenced at a time – the other strand is a complimentary to the sequenced one and as such it’s sequence could be inferred from the DNA sequencing results.
The DNA sequence is too long – millions to billions of nucleotide bases, and must be split up to multiple fragments. Then these DNA sequence fragments must be multiplied and for that purpose are used vectors systems. The vectors are actually cells that indefinitely self-replicate it’s genetic material. Another important factor for the multiplication are the specific primers – particular polynucleotide sequence which is specific for a DNA structure in the targeted sequence. The DNA sequencing reaction needs the primer to start the biochemical reaction of synthesis of the second DNA strand. The sequencing process is performed in a thermal cycler because the reaction needs to be repeated many times. Actually four separate DNA synthesis reactions are running in parallel – each of these separate reactions contains labeled nucleotides for just one of the four bases. The generation of multiple copies of one and the same DNA clone makes possible the analysis of the resulting DNA clones and their ordering in the next step.
After the DNA clones synthesis, they are sorted by their size using capillary electrophoresis – DNA molecules are pulled by an electricity through a gel in a very thin glass tube (capillary). Four different paths are loaded with DNA clones – one for each dye-labeled nucleotides (G, C, A and T). The capillary electrophoresis order DNA strands by their length in an ordered manner. As they emerge from the capillary, they pass through a laser that activates and identifies labeled nucleotide bases in each clone. The labeled bases in the four different DNA synthesis streams are read from the shortest clone to the longest and this information is loaded into a computer, which process the raw results and generates the output of the DNA sequencing.
Practical Applications of DNA Sequencing Technology
DNA sequencing techniques are used to determine the sequence of single genes, large genetic sequences, full chromosomes and even complete genomes. This is useful in various fields of life sciences, like:
- Molecular biology – studying the genome as a whole; identify the coding sequences for proteins; identifying the genes and their variations; unveiling the associations between genes and diseases or phenotypes; suggesting possible drug targets
- Evolutionary biology – studying the onto-genetic relations between different organisms and how they have evolved
- Metagenomics – screening for species present in different biomes like sea water, sewage, soil, debris filtered from the air.
Similar to DNA sequencing, non-sequencing techniques like DNA fingerprinting could provide less-precise but still valuable information that can be useful for:
- Medicine – detect the presence of known genes
- Forensic identification
- Parental testing and genealogy
Basic DNA Sequencing Methods
- Maxam-Gilbert Sequencing – the first developed DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases in the DNA sequence.
- Sanger Sequencing – a DNA sequencing method relies on in vitro DNA replication during which DNA polymerase selective incorporates chain-terminating dideoxynucleotides at different points of the DNA sequence.
Advanced and De Novo DNA Sequencing Methods
- Shotgun Sequencing
- Bridge PCR
Next-generation DNA Sequencing Methods
- Massively parallel signature sequencing (MPSS)
- Ion Torrent sequencing
- Illumina dye sequencing
- SOLiD sequencing
- DNA nanoball sequencing
- Single molecule real time sequencing (SMRT)