What is DNA (Deoxyribonucleic acid) Sequencing?

The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.

Determination of DNA Sequences

There are three methods of DNA sequencing:

• Maxam Gilbert method
• Sanger sequencing method
• Automated DNA sequencing method.

The field of genomics is the study of the molecular organization of the genome, their content information, and all the products of the genes that they encode.

Genomics is divided into three fields of study:

Structural genomics deals with organizing genomic structures within individual genomes.

Functional genomics deals with the study of the function of all gene sequences and their expression in an organism.

Comparative genomics is the study of differences and similarities in the genome structure (DNA or RNA) and organization of different organisms.

DNA Sequencing Methods

• Allan Maxam and Walter Gilbert developed a method of DNA sequencing which is chemical in nature.
• In this, base-specific cleavage reaction occurs at End labeled DNA before its separation in the gel. But this method is not commonly used. The paired ends are subjected to digestion with the help of restriction enzymes.
• In the same year (1977), Fredrick Sanger and co-workers developed an enzymatic method of DNA sequencing known as the dideoxynucleotide chain (Sanger sequencing) termination method that produces a ladder of molecules. Several cell organelles like mitochondria found in Eukaryotes, their mitochondrial DNA can be easily sequenced by sequencing machines.
• There is a need of four types of deoxynucleotide triphosphate (dNTP) used - dATP, dGTP, dCTP, dTTP.
• And four types of dideoxynucleotide triphosphate (ddNTPs) used - ddATP (dideoxyadenine triphosphate), ddTTP (dideoxythymine triphosphate), ddCTP (dideoxycytosine triphosphate), ddGTP (dideoxyguanine triphosphate).

Methods of DNA Sequencing

• The Sanger Sequencing Method is based on the elongation of the DNA chain with the help of an enzyme known as the DNA polymerase enzyme.
• The Maxam Gilbert method is based on the chemical, degradation of long chains of DNA molecule.

Maxam and Gilbert Method

• The first DNA sequencing technique using chemical reagent was developed by Maxam and Gilbert.
• The 5' ends of the ds DNA are replaced by radiolabelled phosphate. This is achieved by using an enzyme known as Polynucleotide kinase.
• After this denaturation process occurs with the help of NaOH that denatures the strands into single strands which are then separated by Electrophoresis. Mechanical digestion selects exons leading to exome sequencing.
• The four tubes were taken which are labeled into which labeled DNA are distributed.
• Out of four tubes, In tubes 1 and 2, dimethyl sulfate which methylates purines is added. Methylation of guanine is 5 times more efficient as compared to adenine.
• This methylation is random in the process that leaves differences in A and G composition in each strand.
• Heating is performed to remove methylated bases leaving only deoxyribose.
• Now, Alkali treatment is given that breaks the sugar-phosphate backbone.
• DNA fragments are produced of varying size and length due to the heat treatment.
• Each fragment is determined by the different positions of methylated guanine and adenine because guanine is methylated more often than adenine. Sample 1 or tube 1 has only the "G" only fragment.
• The tube "2" has not undergone any heat treatment but treated with dilute acid, which removes methylated adenine and some guanine.
• Then, alkali treatment is given that breaks down the sugar-phosphate chain at the place from where Adenine was removed. So. the tube has mixed portions of methylated adenine and guanine. Hence, this tube has A + G fragments.
• The positions of A and G are determined by the following rule:-If the band contains n-nucleotides in both the A+G and G the only lane, then G exists at position n+1 in the original molecule.If the band contains n-nucleotides only in the A+G lane, then A exists at position n+1 in the original molecule.
• In the third tube, the sample is treated with hydrazine that reacts with cytosine and thymine but not with A and G. Then next treatment is given which is of piperidine. This breaks the backbone of sugar-phosphate at the 5' end that has reacted with hydrazine.
• In the fourth tube, hydrazine with NaCl treatment is given that only reacts with cytosine, this is also followed by piperidine treatment which breaks the backbone.
• The positions of C and T are determined by the following rule:
  • If the band contains n-nucleotides in both the C +T and C the only lane, then cytosine exists at position n+1 in the original molecule.
  • If the band contains n-nucleotides only in the C+T lane, then Thymine exists at position n+1 in the original molecule.
• Then all these four samples undergo electrophoresis simultaneously so that all bands appear in a single lane of the gel. The sequence can be read directly from the gel. The complementary band is also sequenced for confirmation.

Now, this gel is exposed to the x-ray film for autoradiography that will give a series of dark bands.

Sanger Method

It is a physical method and a powerful one for DNA sequencing that uses single-stranded DNA as a template. This method is also called the dideoxynucleotide chain termination method. This method is a high throughput sequencing method that is widely used for sequencing DNA with long chains.

Requirements

• DNA Primer
• DNA Polymerase
• Normal deoxynucleoside triphosphates (dNTP's)
• Modified di-dNTP, terminates DNA strand elongation.

If an incoming nucleotide lacked a 3' hydroxyl group and further elongation would be stopped.

Dideoxysugar + Incoming nucleotide → No chain elongation occurs.

• These chain-terminating nucleotides lack a 3' OH group required for the formation of a phosphodiester bond between two nucleotides that will force the DNA polymerase enzyme to cease the extension of DNA when a modified ddNTP is incorporated.
• These ddNTP may be radioactively or fluorescence-based labeled for detection in automated sequencing methods.
• The chromosomes are first cut into pieces to a few hundred base pairs long sizes which can be sequenced easily.
• This Sanger sequencing method can be done even if a small amount of nucleotide is present that contains the sugar deoxyribose instead of deoxyribose.
• For sequencing DNA, four DNA syntheses are carried out in each reaction contains the ssDNA template to be sequenced. Each reaction produces a set of fragments that terminates at the point at which dideoxynucleotide was randomly inserted in the place of the normal deoxynucleotide.
• Therefore, in each of these four reactions, the length of fragments is determined by the position in the daughter strand and also depends on the position of incorporation of ddNTPs.
• After completion of the reaction, the fragments of each of the tubes are separated by electrophoresis into four different lanes of the high resolution of polyacrylamide gel.
• This gel is then dried and autoradiography is performed to reveal the position of different bands.
• The lane contains the fragments with ends that correspond to the ddNTPs used.
• By reading from top to bottom of the gel, the DNA sequence can be read amplified by using Polymerase chain reaction and bands by means of autoradiography of all four lanes.
• This Sanger sequencing method is a next-generation sequencing method that delivers compatible results.

Significance of DNA Sequencing

• This technique is used in sequencing the different DNA sequences that help the researchers in identifying the gene which they are studying that encodes for specific proteins being translated during the process of the central dogma.
• This also helps in identifying a particular gene that is responsible for causing disease or for producing a desired drug.

Context and Applications

This topic is significant in the professional exams for both undergraduate and graduate courses, especially for

• B.Sc Biology
• M.Sc in Microbiology
• Masters in Microbial Biotechnology