DNA sequencing: Definition and FAQs

DNA sequencing 

Have you ever heard of DNA sequencing? For example, human genome sequencing was accomplished in 2003, after years of international efforts. But what does sequencing of a genome or even a small fragment of DNA mean? 

What is DNA sequencing?

DNA sequencing is recognising the sequence of the bases of nucleotides in a chunk of DNA. In other words, DNA sequencing means finding the sequence of the four fundamental building blocks of DNA- known as “bases” – that form the molecule of DNA. The sequence informs the scientists about the kind of genetic information carried in a particular segment of DNA.

Suppose scientists can utilise sequencing information to find out which stretches of DNA carry genes and which stretches contain regulatory instructions used for turning genes on or off. Additionally, and importantly, sequence data can highlight alterations in a gene that may lead to disease.

In the double helix structure of the DNA, the four bases of the DNA always pair with the same one to give “base pairs.” Adenine (A) pairs up with thymine (T), and cytosine (C) pairs up with guanine (G). This pairing is fundamental to the method by which molecules of DNA are replicated when cell division occurs.

The pairing is also fundamental to the methods by which most experiments related to DNA sequencing are performed. The human genome carries billions of base pairs that give out the instructions for creating and maintaining the form of a human being.

Nowadays, with the right equipment and materials, sequencing a short chunk of DNA sequence charts has been made possible. However, sequencing an entire genome (all of the DNA of an organism) remains a challenging task. It involves breaking the DNA sequence chart of the genome into many smaller fragments, sequencing the pieces, and arranging the sequences into a single long “consensus.”

Thanks to modern methods that have been developed over the past few decades, genome sequencing is now much quicker and less expensive than it was during the Human Genome Project.

In this discussion, we will take a look at the methods used for DNA sequencing. We will focus on one reliable method, Sanger sequencing, but we will also discuss new (“next-generation”) methods that have decreased the cost and accelerated the speed of large-scale DNA sequencing.

Sanger sequencing: The termination of chain method

Sanger sequencing is a technique of DNA sequencing that is routinely used to sequence fragments of a DNA sequence chart having about 900 base pairs. It is also called the termination of chain method and was developed in 1977 by the scientist from Britain Fred Sanger and his partners.

The Sanger sequencing method was employed to find out the sequences of several small fragments of DNA of humans in the project of the human genome. These fragments were not necessarily 900 base pairs or lower, but scientists could decipher each fragment with the employment of multiple rounds of Sanger sequencing. The parts of DNA were lined up according to their coinciding parts to decipher the sequences of larger portions of DNA and, finally, the whole chromosome.

Even though now genomes are generally sequenced with other faster and economically favourable methods, still Sanger sequencing is widely used for sequencing individual DNA pieces, like fragments employed in the cloning of DNA or created through PCR.

Ingredients for Sanger sequencing

Sanger sequencing requires creating innumerable replicas of a target region of DNA. It has ingredients that are the same as those required for DNA replication in or for PCR, which replicates DNA in vitro. They include:

• An enzyme called DNA polymerase
• A primer is a short fragment of single-stranded DNA that connects with the template DNA and functions like a beginner for the polymerase.
• The four DNA nucleotides.
• The DNA that has to undergo sequencing
• A reaction of Sanger sequencing also consists of a specific ingredient— Dideoxy, versions of four nucleotides, each labelled with a dye of different colour.

These nucleotides are akin to regular nucleotides, but with one main difference: there is no hydroxyl group on the carbon on the 3′ end of the ring of sugar. In a normal nucleotide, the hydroxyl group on the 3′ end functions as a “hook,” granting access to a new nucleotide that needs to be added to a pre-existing chain.

After a dideoxy nucleotide is added to the chain, no hydroxyl is available, and no further nucleotides can be added. The chain ends with the dideoxy nucleotide, marked with a particular colour of dye based on the base (A, T, C or G) it carries.

Method of Sanger sequencing

The sample of DNA to be sequenced is mixed in a tube with primer, DNA polymerase, and nucleotides. The four dye-labelled, chain-termination dideoxy nucleotides are combined with it as well, but in smaller quantities than the ordinary nucleotides.

Firstly, the mixture is subjected to high temperatures to cause denaturation of the template DNA sequence and detach the strands. Its temperature is then brought down so the primer can bond with the single-stranded template. The temperature is raised again after the primer has bonded, allowing DNA polymerase to produce new DNA starting from the primer.

DNA polymerase keeps adding nucleotides to the chain until a dideoxynucleotide is added instead of a normal one. After that, no further nucleotides can be added, and the strand completes with the dideoxy nucleotide.

This process is repeated over and over again in several cycles. When the cycling is complete, it is practically guaranteed that a dideoxy nucleotide will have been bonded at every position of the target complementary DNA sequence in at least one reaction. That means the tube will have fragments of different lengths, closing at each nucleotide position in the original complementary DNA sequence. The extremities of the fragments will be labelled with dyes that designate their final nucleotide.

Next-generation sequencing

If you are wondering that the name sounds like Star Trek, that is what it is called! The most modern set of DNA sequencing techniques is collectively called next-generation sequencing.

A variety of next-generation sequencing techniques using different technologies are available. However, most of those have common characteristics that differentiate them from Sanger sequencing:

Greatly parallel: several reactions of sequencing occur at the same time

Microscale: reactions are tiny, and many can be performed at once on a chip

Fast: because reactions are performed in parallel, results are ready much quicker

Low-cost: sequencing a genome is less expensive than with Sanger sequencing

Shorter length: reads typically range from 50-700

nucleotides in length

Are any new sequencing technologies being developed?

One new sequencing technology involves observing DNA polymerase molecules as they replicate DNA (the same molecules that make new DNA copies in our cells) with a very fast and efficient movie camera and microscope and integrating different bright dyes of different colours, one each for the letters A, T, C and G. This method gives different and very helpful information than what is supplied by the instrument systems that are used most commonly.

Another modern technology in development involves the use of nanopores for DNA sequencing, which involves threading single strands of DNA sequencer via extremely small membranous pores. The bases of complementary DNA sequences are walked through one at a time as they compress through the nanopore. The bases are pointed out by estimating the differences in their consequence on ions and the current that flows via the pore.

Using nanopores for what DNA profiles utilise DNA sequencing offers lots of potential advantages over the current techniques. The goal is to decrease the cost of sequencing and to do it faster. Unlike current sequencing methods, nanopore DNA profiles utilise DNA sequencing to ensure researchers can study the same molecule repeatedly.

Conclusion 

Although routine DNA profiles utilising DNA sequencing in a doctor’s office is still a far-fetched dream, some large medical centres have started to use sequencing to diagnose and treat some medical conditions. For example, in cancer cases, physicians are progressively using sequencing data to identify the particular cancer type a patient has.

This enables the physician to make better treatment choices. Differentiating the genome sequences of various types of animals and organisms, such as yeast and chimpanzees, can also give insights into the biology of development and evolution.

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