DNA sequencing

The DNA structure consists of four main nucleotide bases such as adenine, guanine, thymine, and cytosine. These nucleotide bases get arranged in a specific order. Thus, it becomes important to determine the order of the nucleotide bases. Recombinant DNA technology involves amplification of a gene using PCR or cloning techniques. The next step involves the determination of the sequence using a DNA sequencing technique. Determining the sequence helps to find out the underlying mutations associated with a specific genotype. It becomes easy to map the human genome and study diseases associated with gene sequence alterations using DNA sequencing techniques. DNA sequencing not only helps in determining the genetic cause of the disease but also aids in the personalized medicine and pharmacogenomics. Although pharmacogenomics relatively new field, it tremendously helps in determining the response of an individual’s genome towards a particular treatment. It becomes easy to locate the restriction sites using DNA sequencing technique. Gilbert and Sanger shared the Nobel prize for finding out this technique. Maxam Gilbert found out chemical sequencing method. Sanger found out an advanced one. The most commonly used method is known as Sanger’s dideoxy method of DNA sequencing.

Image: DNA sequencing

This method helps in sequencing the linear as well as the circular DNA. The first step involves heat treatment for denaturing the DNA into the single-stranded structure. Next step involves annealing the primer to one of the strands. The primer used in the DNA synthesis consists of an oligonucleotide. The DNA synthesized through this step ensures complementarity with the sequence of interest. Each sequencing experiment requires four set of reactions. The components include single-stranded DNA (to be sequenced), oligonucleotide primer, DNA polymerase, nucleotide precursors such as dATP, dCTP, dGTP, dTTP, and a small amount of dideoxynucleotide ddNTP. A dideoxynucleotide is a modified version of deoxynucleotide. Its deoxyribose sugar consists of a 3’-H instead of 3’-OH. Labeling the primers or the precursors helps in determining the newly synthesized DNA. The reactions consisting of the modified deoxynucleotides or the dideoxynucleotides differ from the others. These modified precursors get added in the reaction mixture to about one-hundredth the amount of unmodified precursor. Upon extension of the primer, the DNA polymerase occasionally adds a dideoxynucleotide. After this step, the DNA synthesis stops. Since the dideoxynucleotide does not have a 3’-OH, a new phosphodiester bond cannot form. Hence, every DNA synthesized using this method consists of a dideoxynucleotide at its end.

The gel electrophoresis helps in separating the DNA chains. The DNA bands get revealed using autoradiography. Reading the sequencing ladder from the bottom to the top helps in determining the sequence of the newly synthesized strand.

DNA sequence analysis:

After determining the sequence, they get an entry into the computer databases using sophisticated techniques. The computer programs help in analyzing the restriction sites, homologous sequences, regulatory sequences, and many more. The designing of the computer programs is such that the possible protein coding regions get detected easily. These programs look for the initiator codon in frame with a stop codon. Hence, these programs detect open reading frame. Genes coding a particular protein get identified easily using the DNA sequencing technique. Segments of DNA capable of expressing a particular phenotype also get identified. It is also possible to identify the genes adjacent to the CpG islands, thereby helpful in assessing the disease phenotypes.

Next-Gen Sequencing:

It involves amplification of the cloned segments and rapid sequencing. This process helps in identifying the bases while incorporating them in the nucleotide chain. Every base emits a fluorescent signal thereby simplifying the identification process. It is a highly scalable technology. It gets classified under the high throughput sequencing and involves massively parallel processing. Next-gen sequencing technique helps in sequencing large stretches of the DNA using parallel processing. It also provides a high resolution and specified the view of the bases, gene or the exome. The intensity of the signal helps in measuring the things accurately. Single nucleotide polymorphisms, insertions, deletions, CNVs, and chromosomal aberrations get detected through next-gen sequencing.

Whole genome sequencing:

The entire genome gets analyzed with the help of whole-genome sequencing. The identification of the genes associated with the inherited disorders, cancers, and other conditions becomes relatively easier using whole genome sequencing. Provides a high resolution of the genome and includes variants. Population studies involve the sequencing of large genomes of the animals, plants, and other organisms. Whole genome sequencing works well with the small as well as large genomes.

Other applications of DNA sequencing:

Thousands of diseases follow the Mendelian pattern of inheritance. Identification of the alleles associated with the disease phenotype becomes easy with the help of sequencing techniques. Certain conditions become so complicated that the patient needs the advice of advanced therapeutics such as bone marrow transplantation. The DNA sequencing reports help in decision making and advice. Pharmacogenomics and personalized medicines, though relatively rare fields, prove beneficial for the diagnosis and early treatment of the individual in a highly specific manner. These techniques also depend on the DNA sequences and other intricate details in the genome. Based on the genome constitution of a patient, prescription of a suitable drug becomes easy.

The Human Genome Project got established successfully through sequencing techniques. Without the sequencing techniques, determining the sequences of the large genomes becomes difficult. High throughput sequencing helps in a better understanding of the oncogenes and tumor-inducing genes in the individuals. Scientists worldwide are trying to deal with these genes to study the effects of various environmental factors on the cancer-causing genes. Thus, sequencing plays a very important role in the diagnostics as well as research. The study of the variations in the genetic compositions of the organisms gets a sophisticated approach using high throughput sequencing. Switching on and off the genes also determines cellular processes. They also involve mutations for the same identified through high throughput sequencing. The sequencing techniques combined with automation also advanced in the field of immunology and medicine. Forensics largely involves DNA sequencing technique. Sequencing technique helps in carrying out a large number of experiments in a very short period. Finished chromosome sequences also get detected accurately through high throughput sequencing and whole genome sequencing.

References:
[1] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read
[2] Medical Genetics, Lynn B. Jorde, John C. Carey, Michael J. Bamshad
[3] Essential Genetics, Daniel Hartl

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