What is DNA Sequencing?
DNA sequencing is considered as one of the important process in determining the sequence of nucleotides which are present in the DNA.
Generally, the genomes are which means that smaller pieces of DNA are sequenced.
In today’s scientifically developed state, sequencing of genomes is relatively easier and it is straight forward.
Features of DNA Sequencing
DNA sequencing helps in determining the precise sequence of nucleotides present in the DNA. Before developing the methods of sequencing of DNAs it is difficult and it involves various procedures and it’s not a direct process.
Later after the development of DNA sequencing methods it is very much useful for performing various techniques.
These technologies have a much advantage of carrying our accuracy, sensitivity, with rate of enhanced speed, and it’s with higher flexibility and it is very easy to use.
However, sequencing a whole genome is a difficult task to perform which requires breaking the DNA present in the genome into many of the smaller genomes and to assemble the sequenced genome into a long single chain there are several methods performed in DNA sequencing and they are as follows.
Types of DNA Sequencing
Sanger DNA Sequencing
It is one of the methods of sequencing DNA, which is also known as chain termination method. Here the regions of DNA are routinely sequenced till 900 base pairs in a lengthy manner.
This method was first practised and performed by Fred Sanger, a British biochemist with his colleagues in the year 1977. In this the human genome project, sangar used this method of sequencing to determine the sequences of small fragments of DNA.
The fragments were aligned by overlapping to the base of the portions and assembles the larger sequences of the DNA and also the entire chromosomes present in the cell.
Though there are many methods found for sequencing the genomes, Sangar sequencing is still used widely in sequencing the DNA as individual pieces.
The sequenced pieces are then further used for cloning of DNA or it is used in generating Polymerase Chain reaction.
Materials Required for Sanger DNA Sequencing
This method involves many copies of target DNA sites. The materials needed are similar as of DNA replication processing; and that includes.
A DNA polymerase enzyme
Primer – a short piece of single strand of DNA which binds the template of DNA and thus acts as the starter for polymerase.
Four nucleotides of DNA – dATP, dTTP, dCTP and dGTP.
Unique ingredient – Dideoxy nucleotides.
Steps of Sanger DNA Sequencing
The sample of DNA which has to be sequenced have to be placed in a tube with a primer, DNA nucleotides, DNA polymerase. Along with the four-dye labelled chain-terminating dideoxy Nucleotides in smaller amounts than the other nucleotides.
The prepared mixture is then heated up in a denature template strand which is made up of DNA and it is cooled to bind up the primer to the single stranded template, further again the temperature is raised, allowing the DNA polymerase to synthesise new DNA.
The DNA polymerase will actively add nucleotides to the chain till the end of the strand reaches. This process is repeated continuously until the cycle completes.
At the end the fragments are labelled with dyes which denotes the final nucleotide. After the reaction is completed the fragments are let to run through the tube which contains a matrix of gel for performing gel electrophoresis.
The short fragments are moved quickly and the long fragments are moved very slowly.
However, when these fragments reach the end line laser is illuminated depending upon the coloured dyes. And from the coloured dyes the original piece of DNA can be detected. And the sequence of DNAs is being read in a chromatogram.
Advantages and Limitations of Sanger DNA Sequencing
This is one of the highly performed sequences for long stretches of DNA which have about 900 base pairs.
It is widely used in sequencing the individual DNA that are present in the cell such as plasmids of bacteria or copy of DNA in polymerase chain reaction.
However, this method has many limitations along with.
It cannot be used sufficiently for large scale sequencing projects such as an entire genome.
Next Generation Sequencing
This is one of the most recently introduced method for sequencing the DNA. It includes variety of techniques with different technologies. They are found some kind of different way from sangar sequencing in the following ways.
Many sequencing reactions can be performed here at the same time.
It also involves processing of tiny and minute sequencings at a single time in one chip.
It can also be performed faster and the results are obtained accurately and much faster.
Cheap at Cost
This type of sequencing of genome is faster comparing with other techniques.
It can also read shorter length of nucleotides from 50 to 700 nucleotides.
Generally, this type of sequencing is a type of running a huge number of tiny sanger sequencing reactions parallelly.
DNA Sequencing Citations