What is DNA Sequencing?: Methods and Techniques Explained
Have you ever wondered exactly how scientists are able to read DNA? Stored in the nuclei of cells, DNA molecules are extremely thin—approximately 2 nanometers wide. To put this into perspective, that’s about 40,000 times smaller than the width of a human hair. And though it’s possible to see DNA under a microscope, scientists are unable to make out much detail, and certainly can’t see the individual units which make up DNA.
So how are they able to get to grips with our genes? The answer is DNA sequencing, a process that enables scientists to identify and understand the genetic information carried within a strand of DNA. Not only is it a key technology to the biology, anthropology, and forensics industries, but DNA sequencing is also integral in determining the results of DNA home testing kits in categories including ancestry and health. Read on to learn all there is to know about this groundbreaking procedure.
What is the purpose of DNA sequencing?
DNA sequencing is used to calculate the exact order of the bases in a strand of DNA. The four bases are adenine (A), thymine (T), cytosine (C), and guanine (G), and together they form two base pairs—adenine with thymine, and cytosine with guanine. These strict pairings enable the two DNA chains to bond and coil into its distinctive double helix structure. The base pairs also provide a simple mechanism for replication, whereby the two strands separate, so an enzyme called DNA polymerase can add replacement bases according to the pairing system (A-T and C-G). As a result, both individual strands become DNA molecules identical to the one from which they derived, and this concept is at the heart of DNA sequencing.
The human genome contains approximately 3 billion base pairs, but scientists are currently unable to read an entire genome, or even a single chromosome, which typically contains between 50 million and 300 million of these pairs. Instead, DNA sequencing breaks a strand of DNA down into more manageable chunks of about 500 bases. After determining the order of the four bases in each DNA fragment, the individual sections are reassembled like a jigsaw puzzle in order to recreate the strand in its entirety. Once scientists know the base order of one strand, they can figure out the order of the second thanks to the base pairing system.
How does DNA sequencing work?
Before DNA can be sequenced, it is cut up into smaller pieces, copied multiple times, and added to a mixture which includes free DNA bases, DNA polymerase enzyme, DNA primers (short single strands of DNA required for replication), and ‘terminator bases’. These are modified DNA bases labelled with fluorescent tags corresponding to either adenine, thymine, cytosine, or guanine.
The sequencing reaction begins by heating everything to 96°C in order to separate the DNA into two single strands. Once the temperature is lowered, DNA primers can bind to the strands. The temperature then rises again, and DNA polymerase attaches to a primer and creates a new strand of DNA by binding free bases according to their complementary pairing. This continues until a terminator base is added, stopping any more bases joining before the mixture is heated once more to separate the new DNA strand from the original. This procedure is continuously repeated until the original DNA strand has been divided into sections that differ in length by just one base, thanks to the positions in which the terminator bases have been added.
The DNA segments are separated according to length, via a process called electrophoresis. The segments are placed at one end of an electrophoresis gel, known as agarose, and electrodes are placed at the other. An electrical current is then applied which causes the DNA to move through the gel. The smaller fragments will move faster, so if there was a TACAGT sequence of DNA, the first piece to make it all the way through the gel would be a T, followed by TA, TAC, and so on.
When the segments reach the end of the gel, a laser causes the terminator bases to light up. The colour is detected by a camera and recorded, so if T was blue, A was red, and C was yellow, the machine would record blue, red, yellow. These colours can then be matched to the corresponding bases to reveal the TAC sequence, which will continue to grow base by base until the sequencing process ends.
Once this procedure is complete, genome scientists will be left with a ‘raw’ sequence where all the short DNA sequences are jumbled together. This raw sequence needs to be reassembled by attaching the individual readings in the correct order to create the continuous DNA strand. As the results need to be double-checked and refined in order to reduce any chances of error, the finishing process usually takes longer than the sequencing itself.
Other DNA sequencing methods
British biochemist Fred Sanger and his team at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, enabled scientists to read a genetic code for the first time, with a process called the Chain Termination method. The method—which is also known as Sanger sequencing— was developed in 1977 and acted as the basis for the DNA sequencing process previously outlined. As there were no machines to record the sequence, an X-ray image of the gel, called an autoradiogram, was made. Scientists recorded the DNA base sequence by hand before manually entering it into a computer. As you would expect, this was a long undertaking, typically taking 12 hours to perform the electrophoresis, another 12 to develop the autoradiogram, and many more hours to read the resulting sequence. It would have taken a year to complete a sequence of 20,000 to 50,000 bases, but the automatic sequencing machines used today can read the same sequence in just a few hours.
Usually, lab-gel machines set electrophoresis gel into a space between two glass plates less than half a millimetre apart, and load DNA into one of 96 lanes. However, newer machines called capillary sequencers run DNA through an array of 96 gel-filled capillaries, which are approximately the width of a human hair. These can sequence each piece of DNA in double the time of a slab-gel machine, and are also fully automated, using a robotic arm to place the DNA into the capillaries. These mechanisms also automatically fill the capillaries with gel and clean them between runs, meaning humans only need to spend about 15 minutes a day refilling containers of gel, water, and other solutions. However, as this technology is so new, some laboratories struggle to get capillary machines to work at top efficiency. As a result, most large-scale projects use a combination of slab-gel and capillary machines for DNA sequencing.
There are new sequencing methods being developed, including watching the polymerase enzyme copy DNA using a very fast movie camera and a powerful microscope, while some scientists use nanopores to sequence DNA. This involves threading single DNA strands through pores in a membrane, and reading the bases one at a time by measuring different influences on ions, as well as the electrical current flowing through the pore.
What can DNA sequencing teach us about our genes?
DNA sequencing is used by DNA home testing providers to examine a customer’s autosomal DNA—the 22 out of the 23 chromosomes that do not determine biological sex—and locate single-nucleotide polymorphism (SNPs). The variants in a person’s genetic sequence are compared to those of previous clients that have been stored in a company’s database. By looking at specific SNPs in the client’s DNA against those most commonly associated with certain characteristics in the reference database, home testing kits can be used to provide information across a range of topics.
For example, SNPs determine traits including skin colour and hair texture, and as certain traits are commonly associated with particular populations, these can be used as markers to determine if, for example, somebody has Asian ancestry. Sequencing can also be used to conduct Short Tandem Repeats (STR) testing, which looks at sections of DNA that repeat themselves in a unique and identifiable pattern to establish familial relationships between people. By distinguishing one DNA sample from another and looking at the number of repeated units, STRs can be used to differentiate between individuals. The more genetic similarities there are, the closer the two individuals are related.
DNA sequencing also yields plenty of information regarding a person’s susceptibility to disease. For instance, 23andMe is able to estimate a customer’s risk of genetic mutation, which could lead to illnesses such as Alzheimer’s or Parkinson’s. Studying an individual’s genetic makeup can also highlight whether they are a carrier of inherited diseases, like sickle cell anaemia or cystic fibrosis, or traits like lactose intolerance and hair loss. This process is occasionally used with couple’s looking to conceive, who may want to know the chances of a child being born with any inherited diseases.
DNA sequences can also be applied to animals, such as chimpanzees, providing insight into the development and evolution within species. And for animals a little closer to home, many companies use sequencing to conduct dog DNA tests. This allows pet owners to learn more about the breed of their furry friend, their behaviours, and any health problems they could be predisposed to.