DNA Sequencing- Maxam–Gilbert Method

DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. 

Sequencing an entire genome (all of an organism’s DNA) is complex. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences. New methods have been developed over the past two decades, that make genome sequencing much faster and less expensive

History

Watson and Crick discovered the structure of DNA in the year 1953. In 1964, Richard Holley performed the sequencing of the tRNA as the first attempt to sequence the nucleic acid.

Using the technique of Holley and Walter Fieser, they sequenced the genome of bacteriophage MS2 (RNA sequencing). The sequenced molecules were RNA, yet DNA sequencing was not performed.

In the year 1977, Fredrick Sanger postulated the first method for sequencing the DNA, named a chain termination method.

In the same year, the chemical method of DNA sequencing was explained by Allan Maxam and Walter Gilbert. The genome of bacteriophage X174 was sequenced in the same year using the chemical degradation method.

Because of the lack of automation, both the methods (chemical degradation and chain termination) were tedious and time-consuming.

The first semi-automated DNA method was developed by Lorey and Smith in the year 1986. In 1987, the Applied Biosystem had developed a fully automated machine-controlled DNA sequencing method. After the development of fully automated machines, the era of the 2000s become a golden period for sequencing platforms.

Furthermore, in 1996, Applied Biosystem developed another innovative sequencing platform known as capillary DNA sequencing. After that, the human genome project was completed by using the combination of these methods in the year 2003.

A fast, accurate, reliable, and highly efficient next-generation sequencing platform was developed in the year 2005 by Solexa/Illumina.

Steps in DNA sequencing

●       Sample preparation (DNA extraction)

●       PCR amplification of target sequence

●       Amplicons purification

●       DNA Sequencing

●       Data analysis

Different methods of DNA sequencing:

Various methods of DNA sequencing

●       Maxam and Gilbert method

●       Chain termination method

●       Automated method

●       Pyrosequencing

●       Whole-genome shotgun sequencing method

●       Next-generation sequencing method

 

Two main methods are widely known to be used to sequence DNA:

1. The Chemical Method (also called the Maxam–Gilbert method).
2. The Chain Termination Method (also known as the Sanger dideoxy method).

Maxam–Gilbert technique is chemical cleavage method (depends on the relative chemical liability of different nucleotide bonds) whereas the Sanger method is the chain termination method (interruption of elongation of DNA sequences by incorporating dideoxynucleotides into the sequences)

The chain termination method is the method more usually used because of its speed and simplicity.

Chemical Cleavage Method (Maxam–Gilbert Method)

Maxam–Gilbert Method

Maxam and Gilbert method was developed in 1977. It is also referred to as a chemical cleavage method. The single-stranded DNA is cleaved at the specific location with the help of the chemicals and the fragments of DNA is then run on polyacrylamide gel.

●       DNA extraction is the first step. After that, the DNA is denatured using the heat denaturation method and single-stranded DNA is generated.

●       The phosphate (5’ P) end of the DNA is removed (by alkaline phosphatase enzyme) and radiolabeled with P32 (by polynucleotide kinase).

●       4 different chemicals are used to cleave DNA at four different positions; hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides while dimethyl sulfate and formic acid attack purine nucleotides. The modified DNAs may then be cleaved by hot piperidine

⮚      Hydrazine: T + C

⮚      Hydrazine NaCl: C

⮚      Formic acid: A + G

⮚      Dimethyl sulfate: G

●         An equal volume of 4 different ssDNA samples is taken into 4 different tubes each containing these 4 different chemicals. The samples are incubated for some time and electrophoresed in polyacrylamide gel electrophoresis.

●      A series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule

●      Fragments electrophoresed in polyacrylamide gel electrophoresis for size separation.

●      To visualize the fragments, the gel is exposed to X-ray film for autoradiography (due to radiolabelled 32P end of the DNA-a series of dark bands each showing the location of identical radiolabeled DNA molecules.

●      From presence and absence of certain fragments the sequence may be inferred

Important features

●       Base-specific cleavage of DNA by certain chemicals

●       Four different chemicals, one for each base

●       A set of DNA fragments of different sizes

●       DNA fragments contain up to 500 nucleotides

Advantages

●       Purified DNA can be read directly

●       Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences

●       Can be used to analyze DNA protein interactions (i.e. footprinting)

●       Can be used to analyze nucleic acid structure and epigenetic modifications to DNA

Disadvantages

●       It requires extensive use of hazardous chemicals.

●       It has a relatively complex set up / technical complexity.

●       It is difficult to analyze more than 500 base pairs.

●       The read length decreases from incomplete cleavage reactions.

●       It is difficult to make Maxam-Gilbert sequencing based DNA kits.

However, the method is more accurate than Sanger sequencing. It is more advantageous over the Sanger method because the purified DNA is directly used for sequencing.

It’s best suitable for DNA footprinting and DNA structural studies.  It is used in automated techniques for DNA fingerprinting and genetic engineering studies.