DNA Sequencing: Definition, Methods, Examples
Nucleotides are the chemical building blocks of life and are found in the DNA of living organisms. Each nucleotide consists of a sugar, phosphate and a **nitrogen-containing base**: adenine (A), thymine (T), cytosine (C) and guanine (G). The specific order of these nucleotide bases determines which proteins, enzymes and molecules will be synthesized by the cell.
Determining the order, or the sequence of nucleotides, is important for the study of mutations, evolution, disease progression, genetic testing, forensic investigation and medicine.
Genomics and DNA Sequencing
Genomics is the study of DNA, genes, gene interactions and environmental influences on genes. The secret to unraveling the complex inner workings of genes is being able to identify their structure and location on chromosomes.
The blueprint of living organisms is determined by the order (or sequence) of nucleic acid base pairs in DNA. When DNA replicates, adenine pairs with thymine, and cytosine with guanine; mismatched pairs are considered mutations.
Since the double helix deoxyribonucleic acid (DNA) molecule was conceptualized in 1953, dramatic improvements have been made in the field of genomics and large-scale DNA sequencing. Scientists are diligently working to apply this new knowledge to individualized treatment of diseases.
At the same time, ongoing discussions allow researchers to stay ahead of the ethical implications of such rapidly exploding technologies.
Definition of DNA Sequencing
DNA sequencing is the process of discovering the sequence of various nucleotide bases in snippets of DNA. Whole-gene sequencing allows comparisons of chromosomes and genomes present in the same and different species.
Mapping out chromosomes is useful for scientific research. Analyzing the mechanisms and structure of genes, alleles and chromosomal mutations in DNA molecules suggests new ways of treating genetic disorders and stopping cancerous tumor growth, for instance.
DNA Sequencing: Early Research
Frederick Sanger's DNA sequencing methods greatly advanced the field of genomics starting in the 1970s. Sanger felt ready to tackle DNA sequencing after successfully sequencing RNA when studying insulin. Sanger was not the first scientist to dabble in DNA sequencing. However, his clever DNA sequencing methods – developed in tandem with colleagues Berg and Gilbert – earned a Nobel Prize in 1980.
DNA Sequencing Basics
DNA Sequencing Basics
Sanger knew the potential value of his work and often collaborated with other scientists who shared his interests in DNA, molecular biology and life science.
Although slow and expensive in comparison to today's sequencing technologies, Sanger's DNA sequencing methods were lauded at the time. After trial and error, Sanger found the secret biochemical "recipe" for separating strands of DNA, creating more DNA and identifying the order of nucleotides in a genome.
High-quality materials can be readily purchased for use in laboratory studies:
• DNA polymerase is the enzyme needed to make DNA.
• DNA primer tells the enzyme where to start working on the DNA strand.
• dNTPs are organic molecules made up of
deoxyribose sugar and nucleoside triphosphates – dATP, dGTP, dCTP and dTTP – that assemble proteins
• Chain-terminators are dye-colored nucleotides, also called terminator nucleotides
for each base – A,T, C and G.
Sanger figured out how to cut DNA into small segments using the enzyme DNA polymerase.
He then made more DNA from a template and inserted radioactive tracers in the new DNA to demarcate sections of the separated strands. He also recognized that the enzyme needed a primer that could bond to a specific spot on the template strand. In 1981, Sanger again made history by figuring out the genome of mitochondrial DNA's 16,000 base pairs.
DNA Sequencing Steps
DNA Sequencing Steps
Temperature must be carefully adjusted throughout the sequencing process. First, chemicals are added to a tube and heated to unravel (denature) the double-stranded DNA molecule. Then the temperature is cooled, allowing the primer to bond.
Next, the temperature is raised to encourage optimal DNA polymerase (enzyme) activity.
Polymerase typically uses the normal nucleotides available, which are added at a higher concentration. When polymerase gets to a "chain terminating" dye-linked nucleotide, the polymerase stops, and the chain ends there, which explains why the dyed nucleotides are called "chain terminating" or "terminators."
The process continues many, many times. Eventually, the dye-linked nucleotide has been placed at every single position of the DNA sequence. Gel electrophoresis and computer programs can then identify the dye colors on each of the DNA strands and figure out the entire sequence of DNA based on the dye, the position of the dye and the length of the strands.
High-throughput sequencing – generally referred to as next-generation sequencing – uses new advancements and technologies to sequence nucleotide bases more quickly and cheaply than ever before. A DNA-sequencing machine can easily handle large-scale stretches of DNA. In fact, the entire genomes can be done in a matter of hours, instead of years with Sanger's sequencing techniques.
Next-generation sequencing methods can handle high-volume DNA analysis without the added step of amplification or cloning to get enough DNA for sequencing. DNA-sequencing machines run multiple sequencing reactions at one time, which is cheaper and faster.
Essentially, the new DNA sequencing technology runs hundreds of Sanger reactions on a small, easily readable microchip that is then run through a computer program that assembles the sequence.
The technique reads shorter DNA fragments, but it is still faster and more efficient than Sanger's sequencing methods, so even large-scale projects can be quickly completed.
The Human Genome Project, completed in 2003, is one of the most famous sequencing studies done to date. According to a 2018 article in Science News, the human genome consists of approximately 46,831 genes, which was a formidable challenge to sequence. Top scientists from around the world spent almost 10 years collaborating and consulting. Led by the National Human Genome Research
Institute, the project successfully mapped out the human genome using a composite sample taken from anonymous blood donors.
The Human Genome Project relied on bacterial artificial chromosome (BAC-based) sequencing methods to map out base pairs. The technique used bacteria to clone DNA fragments, resulting in large quantities of DNA for sequencing. The clones were then reduced in size, placed in a sequencing machine and assembled into stretches representing human DNA.
New discoveries in genomics are profoundly changing approaches to disease prevention, detection and treatment. The government has committed billions of dollars to DNA research. Law enforcement relies on DNA analysis to solve cases. DNA testing kits can be purchased for home use to research ancestry and identify gene variants that may pose health risks:
• Genomic analysis entails comparing
and contrasting the genome sequences of many different species in the domains
and kingdoms of life. DNA sequencing can reveal genetic patterns that shed new
light on when certain sequences were introduced evolutionarily. Ancestry and
migration can be traced via DNA analysis and compared to historic records.
• Advances in medicine are happening at an exponential rate because virtually
every human disease has a genetic component. DNA sequencing helps scientists
and doctors understand how multiple genes interact with each other and the
environment. Quickly sequencing the DNA of a new microbe causing a disease outbreak
can help identify effective medicines and vaccines before the problem becomes a
serious public health issue. Gene variants in cancer cells and tumors could be sequenced
and used to develop individualized gene therapies.
• Forensic science applications have been
used to help law enforcement crack thousands of difficult cases since the late
1980s, according to the National Institute of Justice. Crime scene evidence may contain samples of DNA from
bone, hair or body tissue that can be compared to the DNA profile of a suspect
to help determine guilt or innocence. The polymerase chain reaction (PCR) is a
commonly used method to make copies of DNA from trace evidence prior to
sequencing.
• Sequencing newly discovered species can help identify
which other species are most closely related and reveal information about
evolution. Taxonomists use DNA "barcodes" to classify organisms. According to the
University of Georgia in May 2018, there are an estimated 303 species of
mammals yet to be discovered.
• Genetic testing for diseases look for mutated gene
variants. Most are single nucleotide polymorphisms (SNPs), which means only one
nucleotide in the sequence is changed from the "normal" version. Environmental
factors and lifestyle affect how and if certain genes are expressed. Global
companies make cutting-edge new-generation sequencing
technologies available to researchers around the world interested in multigene
interactions and whole-genome sequencing.
• Genealogy DNA kits use DNA sequences in
their database to check for variants in an individual's genes. The kit requires
a saliva sample or cheek swab that is mailed to a commercial lab for analysis.
In addition to ancestry information, some kits can identify single nucleotide
polymorphisms (SNPs) or other well-known genetic variants such as the BRCA1 and
BRCA2 genes associated with elevated risk for female breast and ovarian cancer.
New technologies often come with the possibility of social benefit, as well as harm; examples includes malfunctioning nuclear power plants and nuclear weapons of mass destruction. DNA technologies come with risks, too.
Emotional concerns about DNA sequencing and gene-editing tools like CRISPR include fears that the technology might facilitate human cloning, or lead to mutant transgenic animals created by a rogue scientist.
More often, ethical issues related to DNA sequencing have to do with informed consent. Easy access to direct-to-consumer DNA testing means consumers may not fully understand how their genetic information will be used, stored and shared. Lay people may not be emotionally ready to learn about their defective gene variants and health risks.
Third parties such as employers and insurance companies could potentially discriminate against individuals who carry defective genes that may give rise to serious medical problems.
References
- Genomics: The Sequence of Sequencers: The History of Sequencing DNA
- What Is Biotechnology: The Ultimate Goal: Sequencing DNA
- Science News: A Recount of Human Genes Ups the Number to at Least 46,831
- University of Georgia: Scientists Predict Number of Undiscovered Mammal Species
- National Cancer Institute: BRCA Mutations: Cancer Risk and Genetic Testing
- National Institute of Justice: Forensic DNA
- YourGenome.org: What Is PCR (Polymerase Chain Reaction)?
Cite This Article
MLA
Dowd, Mary. "DNA Sequencing: Definition, Methods, Examples" sciencing.com, https://www.sciencing.com/dna-sequencing-definition-methods-examples-13718447/. 29 May 2019.
APA
Dowd, Mary. (2019, May 29). DNA Sequencing: Definition, Methods, Examples. sciencing.com. Retrieved from https://www.sciencing.com/dna-sequencing-definition-methods-examples-13718447/
Chicago
Dowd, Mary. DNA Sequencing: Definition, Methods, Examples last modified August 30, 2022. https://www.sciencing.com/dna-sequencing-definition-methods-examples-13718447/