By: Freya Preimsberger
According to one sequencing expert, DNA sequencing and the information it brings are well on their way to being ubiquitous in the field of food safety. Whole-genome sequencing, which determines the DNA sequence of a particular organism’s entire genome, has already been used in monitoring foodborne disease outbreaks, in determining the species of microorganisms in a sample and in characterizing antibiotic resistant bacteria. Genotypic typing has proved more effective at discriminating different species of microbes, such as those in the genus Salmonella, when compared to traditional phenotypic typing. As scientists make developments in sequencing technology, the costs associated with its use decrease and the technology becomes more accessible to those in food safety and other fields. It’s important for people in the field of food safety to have a basic understanding of DNA sequencing and how to use it to their advantage.
What is DNA?
DNA, or deoxyribonucleic acid, is a molecule containing hereditary material and is present in all cells and some viruses. In 1953, scientists James Watson and Francis Crick discovered that DNA is in the shape of a double helix, or a long twisted ladder. Two strands, each made up of nucleotides, wind around each other. Each nucleotide consists of a sugar called deoxyribose, a phosphate group and one of four nitrogenous bases: adenine, guanine, cytosine or thymine. Covalent bonds form between phosphate groups and sugars in a single strand, making up the sugar-phosphate backbone. The double helix forms because of hydrogen bonding between complementary nitrogenous bases – normally, adenine bonds with only thymine and guanine bonds with only cytosine. The linear sequence of A, G, C or T is what encodes genetic information. The base-pairing of DNA is what enables processes like replication and gene expression. Cells use DNA for transcription, where one strand of DNA is used to synthesize RNA, or ribonucleic acid, and translation, where RNA is used to make proteins.
After the discovery of DNA’s structure, other advancements in the field laid the foundation for the development of DNA sequencing. Restriction enzymes, for example, can cut a sequence at a specific spot, fragmenting a long strand of DNA into smaller ones. The enzyme DNA polymerase can add complementary nucleotides to a single strand of DNA by using deoxyribonucleotide triphosphates, which are a reactive form of the nucleotide monomers found in DNA. In 1983, Kary Mullis developed polymerase chain reaction, which allows for the amplification of a piece of DNA into millions of copies. A sample of DNA is heated until its two strands separate. Primers, nucleotides and Taq DNA polymerase are added, filling in a complementary strand for each of the two strands that were separated. This process, which doubles the amount of DNA in each round, can be repeated until a large number of copies have been made. The technique has since become an essential tool in the field of molecular biology, and Mullis was awarded the Nobel Prize in Chemistry in 1993 for his work. Another important development in the field of genomics is Sanger sequencing, also known as the chain termination method. A DNA sample of appropriate length is combined with a primer, DNA polymerase, regular deoxyribonucleotide triphosphates and dideoxyribonucleotide triphosphates, or ddNTPs; these modified ddNTPs differ from the usual ones in that, once one is added to the growing DNA strand, DNA polymerase cannot add any more nucleotides. They also contain fluorescent tags differentiating between each base. Replication of the new strand stops at each ddNTP, making many partial copies of the parent strand of different lengths. The many strands can then be sorted by increasing size with gel electrophoresis. By reading the tags on the ddNTPs as the strands get longer, the DNA sequence of the original strand can be determined. Sanger sequencing was the go-to for DNA sequencing for many years, but it was limiting in that it can only sequence short DNA fragments with lengths less than 1000 nucleotides.
DNA and Mapping
In 1990, scientists from around the world established the Human Genome Project with the aim of sequencing the entire human genome and mapping all of its genes. Sanger sequencing proved to be prohibitively inefficient and very expensive for sequencing all three billion base pairs in the human genome. The project created a demand for faster and cheaper sequencing technology. Strides in biological techniques eventually lead to the development of a number of new sequencing methods grouped together as next-generation sequencing, or NGS. Millions of DNA strands can be sequenced in parallel, and fragments are pieced together by computer software to produce whole genomes. NGS is much faster – the entire human genome can be sequenced in a day – and cheaper than Sanger sequencing. It can also more effectively detect different types of mutations.
Although NGS encompasses several different high throughput sequencing techniques, they all have the same basic steps: library preparation, sequencing and data analysis. During library preparation, the DNA sequence is randomly split into small fragments. Sequences known as adapters are added to immobilize and anchor the DNA fragments to support structures. The “library” refers to the fragments to be sequenced that are held by the adapters. These pieces are then amplified, often through polymerase chain reaction. The second step, sequencing, depends on the type of NGS being used. However, they all use less reagent than Sanger sequencing does, keeping costs low. In the last step, computer software puts the sequences of the fragments in order.
In the field of microbiology, NGS is used to characterize pathogenic microorganisms and acts as an improved replacement for the phenotypic typing previously used. Genotypic typing of microorganisms through genome sequencing provides information on what species it is and potential antibiotic resistance. Mapping the genomes of different microorganisms may shed light on their relationship and point to the source of an outbreak. A juncture from phenotypic to genotypic typing can be seen at the creation of PulseNet by the Centers for Disease Control and Prevention in 1996. PulseNet is a network that, with health and food agency laboratories, fingerprints subtypes of Escherichia coli and other bacteria that cause foodborne illness. The network has established a standard method for subtyping bacteria and been extremely effective in detecting and observing outbreaks from foodborne pathogens. New sequencing technologies, such as whole-genome sequencing, are soon to be used in foodborne disease outbreak surveillance. Advances to come in genomics technology and bioinformatics have the potential to change how food safety is monitored, leading to advancements in food safety.