DNA Sequencing is a way to figure out the order of nucleotides along a strand of DNA. The four basic nucleotides are adenine (A), guanine (G), cytosine (C), and thymine (T). These nucleotides are the chemical building blocks of DNA. The order, or sequence, of these blocks tells your cells how to behave.
In the early 1980s, scientists discovered two nucleotides that had slightly modified chemical structures. Today, we know of four modified nucleotides. These are methyl-cytosine (mC), methyl-adenine (mA) and two other variations of cytosine (5-formylcytosine and 5-carboxylcytosine). The functions of these modified nucleotides are still being studied. So far, scientists have found that they play an important role in switching genes on and off. One example is the inactivation of the X chromosome.
Did you know?
In 2019, a team of scientists led by Shuichi Hoshika added four synthetic nucleotides to the four natural ones. This produced an eight-letter genetic code that can be used to generate hachimoji DNA.
All humans have 99.9% identical DNA. But the other 0.1% results in a lot of things that make each of us unique. These are things like hair colour or blood type. Even whether or not you like the taste of broccoli!
DNA sequencing has become very important to the fields of medicine, biotechnology, forensics and many others. This technology can help scientists find the genes involved in diseases and work towards cures. DNA sequencing can also help scientists investigate crimes. This is done using DNA evidence like hair or skin cells.
History of DNA Sequencing
In the late 1970s, scientists developed the first two methods of DNA sequencing. Allan Maxam and Walter Gilbert developed Maxam-Gilbert sequencing. This is also called chemical sequencing. Their method used chemicals to break DNA into small chunks to determine its sequence. This method was revolutionary at the time, but it is not efficient compared to newer methods.
In 1977, Frederick Sanger and his colleagues developed another method for sequencing DNA. Their method was the most widely used for about 40 years. Even though there are faster and cheaper methods today, the Sanger method is still used a lot.
Did you know?
Frederick Sanger won the Nobel Prize in Chemistry twice! One prize was for his work on the structure of insulin and one was for his DNA sequencing method.
Sanger sequencing is based on the process of DNA replication. Scientists make copies of DNA strands. Then they observe which nucleotides are added. This way the sequence of nucleotides can be seen.
How does Sanger Sequencing Work?
First, you extract a piece of DNA that you want to sequence. Then you heat it. This causes the DNA to unwind. The two strands of the double helix separate into single strands.
Next, you lower the temperature and add a DNA primer. DNA primer is a short sequence of single-stranded DNA. It binds to the DNA strand. Like a starting line, it marks where the sequencing will begin.
Now you raise the temperature slightly. Then you add free nucleotides and an enzyme called DNA polymerase. Free nucleotides contain one of the four bases: cytosine, thymine, adenine or guanine. DNA polymerase begins at the primer sequence and builds a complementary, or opposite, DNA strand. It does this by adding one nucleotide at a time.
Four different sequencing reactions need to take place. One for each of the four different nucleotides. To get these reactions, chemically-altered, chain-terminating versions of each of the nucleotides are added to the mix of free nucleotides. Each of these special nucleotides is labelled with a different colour dye. This allows scientists to see them when they are exposed to UV light.
When the DNA polymerase reaches a chain-terminating nucleotide, it stops the DNA sequence that it was building. The DNA polymerase adds the modified nucleotides randomly. So, many sequences of DNA of different lengths are produced as a result.
Next, the DNA segments undergo gel electrophoresis. Gel electrophoresis is a method that separates pieces of DNA that are different lengths. You add the pieces of DNA to a gel base and expose it to an electric current. This causes the segments to travel through the gel according to their size. The smallest pieces can move the farthest and the largest pieces do not move far at all. Once the pieces have finished moving, the gel is placed under an X-ray or a UV light. This allows scientists to see each segment.
To read the gel, you look at the dark bands in each column. There is one column for each type of nucleotide (G, C, A, T). By reading the sequence of the bands, you can determine the sequence of nucleotides.
Scientists have made changes to the original Sanger method. They use fluorophores. These are small chemical compounds that give off coloured light. A different coloured fluorophore is added to each nucleotide. This way, sequencing can be performed in a single reaction mixture in a capillary tube.
To identify the sequence in the tube, scientists shine a laser through it. When the light passes through each band of colour, a detector creates a peak on a graph. These peaks correspond to the nucleotide bases. By using fluorophores, scientists can automate the process of DNA sequencing. This means it can be done much more quickly.
The Sanger method has allowed scientists to sequence the DNA of many of organisms, from bacteria to humans. Using fluorophores and computers, a person’s DNA (all 3 billion bases) can be sequenced in a few days. Pretty good compared to the years it took previously!
Did you know?
Scientists need to create and use special processes to detect the four new modified nucleotides. This research is important because modifications have roles in development, and in diseases like cancer.