DNA sequencing is the process of determining a nucleic acid sequence in a piece of DNA.
Sequence information can be used to determine genetic information and as a regulatory function to turn genes on and off. DNA sequencing can also be used to find changes in a gene that may cause disease. Two methods are popular for DNA sequencing; they are Sanger sequencing and next-generation sequencing.
Sanger Sequencing
Sanger sequencing is widely used to sequence individual pieces of DNA in cloning through polymerase chain reaction (PCR). Sanger sequencing involves making copies of a targeted DNA region. The ingredients used for Sanger sequencing are; a DNA polymerase enzyme, a primer, four DNA nucleotides (dATP, dTTP, dCTP, dGTP), and the template DNA that is going to be sequenced. Dideoxy-nucleotides differ from deoxy-nucleotides because they lack a hydroxyl group on the 3′ carbon of the sugar ring. This prevents a new nucleotide from being added to an existing chain. The replicated chain ends with a dideoxynucleotide and is marked with a particular dye depending on the base that it carries.
The Sanger method occurs in the following steps. The DNA sample that is to be sequenced is combined in a tube with the primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The chain-terminating dideoxy-nucleotides are added as well. The mixture is first heated to denature the template DNA; then it is cooled, so the primer binds to the single-stranded template. After the primer is bound, the temperature is raised to allow DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase stops adding nucleotides to the chain once it adds a dideoxy-nucleotide. This process is repeated several cycles. Once it is complete, the ends of the fragments will be labelled with dyes that indicate their final nucleotide.
The fragments are then run through a gel matrix and separated using capillary gel electrophoresis. The fragments are illuminated by a laser to detect the attached dye. The smallest fragment crosses through the gel first, and the color of the nucleotide is detected by the laser. Then the next-smallest fragment crosses and the nucleotide is also detected by the laser. The sequence of the DNA is built up one nucleotide at a time. The DNA sequence is read from the peaks recorded by fluorescence intensity shown in a chromatogram. The Sanger sequencing can be used on DNA up to about 900 base pairs and is commonly used to sequence bacterial plasmids or DNA copied in PCR.
Next-generation Sequencing
Next-generation sequencing refers to methods of DNA sequencing developed after the Sanger sequencing method. There are numerous next-generation sequencing techniques that have several features that distinguish them from Sanger sequencing. They are highly parallel, microscale, fast, low-cost, and the DNA sequences are shorter in length (50-700 nucleotides). The ability to routinely sequence genomes quickly and at low cost opens opportunities for personalized medicine based on an individual’s needs.
Practice Questions
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Key Points
• Sanger sequencing uses DNA up to 900 base pairs and commonly used to sequence bacterial plasmids
• The ingredients used for Sanger sequencing are a DNA polymerase enzyme, a primer, four DNA nucleotides (dATP, dTTP, dCTP, dGTP), and the template DNA that is going to be sequenced
• Dideoxy-nucleotides differ from deoxy-nucleotides because they lack a hydroxyl group on the 3′ carbon of the sugar ring
• The difference between next-generation sequencing and the Sanger method is next-generation is highly parallel, microscale, fast, low-cost, and the DNA sequences are 50-700 nucleotides
Key Terms
highly parallel: many sequencing reactions take place at the same time
capillary gel electrophoresis: is an analytical separation method where charged molecules are separated in capillaries filled with a porous gel matrix
sanger sequencing: a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
PCR: Polymerase chain reaction is a method used copies of a specific DNA sample rapidly
primer: a short nucleic acid sequence that provides a starting point for DNA synthesis
DNA polymerase: an enzyme that produces DNA molecules from its building blocks – nucleotides.
Dideoxy-nucleotides: chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing.