What is DNA (Gene) Sequencing?

The structure of DNA (e.g. gene insert, a recombinant plasmid or entire genome) can be analysed by determining the nucleotide sequences. In molecular cloning, the information of nucleotide sequences is essential.

In 1965, Robert Holley and his research group at Cornell University completely sequenced nucleotides of tRNA²*³ (tRNA for yeast alanine).

In 1977, the following two methods were developed. Allan Maxam and Walter Gilbert developed a chemical method of DNA sequencing. In this method, end-labelled DNA is subjected to base specific cleavage reaction before gel separation. In routine sequencing of DNA this method is not commonly followed.

In the same year (1977) Frederick Sanger and co-workers developed an enzymatic method of DNA sequencing. It is also called dideoxynucleotide chain termination method because dideoxynucleotides are used as chain terminator to produce a ladder of molecules.

1. Maxam and Gilbert’s Chemical Dedradation Method :

In this method DNA sequencing involves the following steps:

i. Labelling of 3′ ends of DNA with isotopic phosphorus (³²P).

ii. Separation of two strands labelled at 3′ ends.

iii. Separation of mixture in four sets, each treated with a different reagent which can degrade only G or C, Ar A and G or T and C.

iv. Electrophoretic separation of each sample in four different gel.

v. Autoradiography of gels and determination of the sequence from position of bands in four lanes of gel.

2. Sanger’s (Dideoxynucleotide Chain Termination) Method :

Earlier DNA replication (synthesis) has been described in A Textbook of Biotechnology for Class XI (Chapter 8). The requirements are: a primer with free 3′-OH ends to start DNA synthesis, DNA polymerase and dNTPs. Twice Nobel Prize winner Frederick Sanger developed a powerful method utilizing single stranded DNA as template.

Fig. 3.20 shows the presence of free 3′-OH group at 3′ end in each dATP and no 3′-OH in ddATP. In ddATP a hydrogen atom is attached at 3′ carbon instead of -OH hydroxyl group. If any of four ddNTPs binds the chain elongation is terminated. Because ddNTPs do not have free 3′-OH end which is required for chain elongation. Therefore, no phosphodiester bond will be formed.

Four reaction tubes are labelled with A, T, G and C each containing single stranded DNA template (obtained by NaOH hydrolysis), 5′-radiolabelled DNA primer, and all four radiolabeled dNTPs (dATP, dGTP, dCTP and dTTP) (Fig. 3.21). A small amount of ddATP is added to tube A, ddTTP to tube T, ddGTP to tube G and ddCTP to tube C.

The concentration of ddNTPs should be maintained to about 1 % of the concentration of dNTPs. DNA polymerase is added to each tube, DNA synthesis starts and chain elongates. In each tube ddNTP is randomly incorporated and fragments are terminated. The length of each fragment depends on the position of incorporation of ddNTPs.

After completion of reaction, the fragments of each tube are separated by electrophoresis in four different lanes of high resolution polyacrylamide gel. Then the gel is dried and autoradiography is done so that position of different bands (having radio labeled send) in each lane is observed.

In each lane the ends of fragments contain the base in correspondence to the ddNTPs used. DNA sequence is obtained by reading (from bottom to top of gel) the bands on autoradiogram of four

Automatic DNA Sequencers:

Automatic sequencing machines were developed during 1990s. It is an improvement of Sanger’s method. In this new method a different fluorescent dye is tagged to the ddNTPs. Using this technique a DNA sequence containing thousands of nucleotides can be determined in a few hours.

Each dideoxynucleotide is linked with a fluorescent dye that imparts different colours to all the fragments terminating in that nucleotide.

All four labelled ddNTPs are added to a single capillary tube. It is a refinement of gel electrophoresis which separates fastly. DNA fragments of different colours are separated by their respective size in a single electrophoretic gel.

A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end. The small sized DNA fragments migrate faster and vice versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam.

Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special ‘recorder’. The DNA sequences are read by determining the sequence of the colours emitted from specific peaks as they pass the detector.

This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer. Colour of each dye represents the different nucleotides. Computer converts the data of emitted light in the nucleotide sequences.

DNA sequencing is easier than RNA sequencing due to a greater stability. But RNA sequencing sometimes becomes necessary when one has to determine the position of modified nucleotides present in RNAs.

It is achieved by cleaving 5′ end labelled RNA by using RNases. RNases cleave a particular nucleotide at 3′ end. RNase T1 cleaves after G RNase U-, after A, RNase PhyM after A and U, and Bacillus cereus RNase after U and C. The fragments are analysed by PAGE.