The process which is used for the determination of the sequence of nucleotides (Cs, Gs, As, and Ts) in a DNA piece is called DNA sequencing.
This may include any sort of method or even technology that is used for the determination of the order of four bases (Thymine, Adenine, Guanine and Cytosine). The fast speed of sequencing obtained with modern DNA sequencing technology has been involved in the sequencing of absolute DNA sequences.
The 1st DNA sequences were acquired at the start of 1970s by researchers using laboratory methods based on two-dimensional chromatography.
In this unique article, we will look at two methods that are used for DNA sequencing. We will focus on one deep-rooted method, Sanger sequencing.
Sanger Sequencing: (Chain Termination Method)
DNA regions which are about 900 base pairs sequenced in length using method called Sanger sequencing (Chain termination method). This method was evolved in 1977 by Fred Sanger-British biochemist- and his colleagues.
Sanger sequencing was used for the determination of sequencing of comparatively small fragments of homo-sapiens DNA. The fragments were lined up on overlapping parts to muster the sequences of a larger area of DNA and, in the end, whole chromosomes.
Now genomes are sequenced using different methods that are speedy and inexpensive, but Sanger sequencing is quiet in vast use for the sequencing of single parts of DNA, e.g. fragments used in DNA cloning or fragments formed through polymerase chain reaction (PCR).
Constituents of Sanger Method
Sanger sequencing entails making a large number of copies of selected DNA part. Its constituents are alike to those required for DNA replication in an individual, or for PCR, that copies DNA in vitro.
- The constituents included are:
- An enzyme named DNA polymerase.
- A primer (a small part of single-standard DNA that cohere with template DNA. It works as “starter” for the enzyme polymerase).
- Four types of DNA nucleotides (dGTP, dATP, dCTP,dTTP)
- A Template DNA which is to be sequenced.
Though, the reaction of Sanger sequencing also carry a unique constituent:
Dideoxy (chain-terminating) sort of all 4 nucleotides (ddGTP, ddATP, ddCTP, ddTTP), each one tagged with a separate color of dye.
Dideoxy nucleotides are alike to structured, or deoxynucleotides, but there is one main difference: they have no hydroxyl group on the 3′ C of the sugar ring. In a structured nucleotide, the 3′ hydroxyl group work as a “holder,” permitting anew nucleotide to be attached to an existing chain.
At once the dideoxynucleotide has been attached to the chain then no hydroxyl group available and no other nucleotides can be attached. The chain finishes with dideoxynucleotide, which is pointed out with a specific colour of dye depending on the base (G, A, C or T) which it carries.
Procedure of Sanger Sequencing
- First of all, a DNA sample is taken for whom sequencing required. Then that sample is bonded with DNA polymerase, DNA nucleotides and pa primer. 4 dye-labelled, dideoxynucleotides (chain terminator) are also added in very smaller quantity than other nucleotides.
- First heat the mixture for denaturation of template DNA then cool the mixture for the binding of primer to the single-stranded template. After the binding of primer, the temperature of the mixture becomes high and allow enzyme (DNA polymerase) to form new DNA starting from the primer. Enzymes (DNA polymerase) continue to add nucleotides until it adds dideoxynucleotide in place of normal nucleotides. The Chain terminates at that point and no new nucleotides are added.
- This process is redone in several cycles. After completion of cycles, it’s assumed that dideoxynucleotide is fused at every single point of the target DNA in one reaction. That means, the tube contains different length fragments at every nucleotide point in the original DNA. To indicate their final points the fragment ends are labelled with dye.
- After completing the reaction, the DNA fragments are put in a thin and long tube which contains gel matrix, the process is called Capillary gel electrophoresis. The fragments which are short move quickly and which are long move slowly through gel pores. Each fragment crosses the “end line” at the endpoint of the tube it’s lit up by a laser and allows the combined dye to detect.
The sequence of the crossing of fragments is like that the smallest one in which the primer attaches only one nucleotide only crosses first then next one which contains two nucleotides crosses the end line and so on……. From the dye’s colours that are registered on the detector, the original DNA piece sequence could be built one nucleotide at one time. Data which detector records, contains peak series in fluorescence intensity. The sequence of DNA is read through peaks in the chromatogram.
Some kind of Uses of Sanger Sequencing
High-quality sequencing obtained from Sanger Sequencing for long DNA stretches.
It is used for sequencing of a single piece of DNA, e.g. Bacterial plasmids or DNA copied in PCR.
Some limitations of Sanger sequencing
Sanger sequencing is quite expensive and it is not enough for large projects, e.g. sequencing of the metagenome. Meta genome is the collection of genomes of the microbial community.
For such tasks, new and large scale techniques of sequencing are present which are less expensive and faster.