The generation DNA sequencing instruments generate reads with lengths

The first tactics developed to ascertain the sequence of polynucleotides were not applicable to nucleic acid analysis:  molecules of DNA are significantly longer and are comprised of a small number of nearly identical units, making it difficult to differentiate between them. All studies were carried out on RNA strands, hence new strategies needed to be developed. In 1977 Allan Maxam and Walter presented a DNA sequencing method (known as chemical sequencing) consisting of cleavage at particular bases after chemical modification of DNA. The sanger (chain termination) sequencing method quickly became a the most popular method of choice as after improvements were made in comparison to the Maxam technique it was straight forward reliable and used low amounts of toxic chemicals and radioactivity. Several developments were implemented on Sanger sequencing in the subsequent years, that predominantly included, substitution of phosphor -or tritium – radio-labelling for fluorometric based analysis. Enabling the reaction to happen in one vial rather than in four and enhanced detection by using capillary electrophoresis as an alternative to polyacrylamide gel.  These two improvements influenced the advancement of highly automated sequencing machines (30)(33).  First generation DNA sequencing instruments generate reads with lengths no greater than one kilobase (1kb). Therefor in order to inspect lengthier fragments researches utilized techniques such as ‘shot gun sequencing’, a process where superimposed DNA fragments were replicated and sequenced singly, before being arranged as one long continuous sequence in silicone. The development of the polymerase chain reaction (PCR) and recombinant DNA technologies advanced the genomics revolution by facilitating the production of pure DNA in high concentrations required for sequencing. Less direct routes to improve sequencing also occurred. For instance, a fragment of Escherichicoli DNA polymerase (known as the Klenow fragment lacks 5′- 3′ exonuclease activity) had initially been utilized for sequencing for its ability to incorporate ddNTPs adequately. Nevertheless, more sequenced genomes and methods for genetic control provided resources to discover polymerases that better accommodated the additional chemical moieties of increasingly modified dNTPS .Finally, the Abi prism range, newer dideoxy sequences were introduced which enabled simultaneous sequencing of over two hundred samples.  Coupled with the advancement of mass ‘chain termination’ sequencing research, an additional method arose which in turn lay the foundations for the elementary ground work in the second generation of DNA sequencers. This technique was considerably different from the existing methods, as it does not derive a nucleotides identity via fluorescents or radio labelled dNTPs (or ogligonucleotides) prior to visualizing with electrophoresis. Alternatively researchers used a newly invented luminescent technique used to measure the synthesis of phosphate; this involved a double -enzyme process shown in  DIAGRAM! This approach   regardless of the differences, this pyrosequencing method and Sayen dideoxy are noted as ‘sequence -by -synthesis ‘ (SBS) techniques, as direct action of DNA polymerase to generate visible yield is required. (Contrary to the chemical sequencuing technique). Pal Nyren was the lead pioneer of the pyrosequencing technique. The technique possessed several qualities beneficial to researchers: it could be executed using unrefined nucleotides ( in contrast to heavily- modified dNTPs utilized  in the chain procedures),and surveyed in real time ( in comparison to using lengthy electrophoresis) 51.   Subsequently, advancements included adhering paramagnetic beads to the DNA, and degrading detached dNTPs enzymatically to eliminate lengthy washing steps.  Determining the numbers of identical nucleotides in a row at a particular position was a major difficultly presented by this technique; as the intensity of light discharged is proportional to the magnitude of the homopolymer, however noise generated non-linear data reads over rows of four or five consising of the same nucleotide.  Later pyrosequencing was licensed to biotechnology company, 454 life science. Where is advanced into the first significant ‘next generation technology’.  explain illumia machine in 3 sentnces    There is great debate on what characterizes varying generations of DNA sequencing technology, especially concerning the distinction between second and third. 76. Arguments brought forward are that single molecule sequencing (SMS), simple divergence and real-time divergence from prior technologies should be defining traits. Although is also reasonable that particular technologies can be classified as second/third generation. In this review we define third generation molecules as able to sequence individual molecules, revoking the stipulation for DNA amplification shared by all precedent technologies.  The first SMS technology developed, worked In a similar fashion in which the second-generation IIlumina does, but in the absence of bridge amplification; templates of DNA adhere to a flat surface and the propriety fluorescent reversible terminator dNTPs are used to wash each base individually, next the bases are imaged, cleaved then following base is cycled. This technology was somewhat slow and deemed expensive as relatively short reads were produced, but was the first technology to grant sequencing of non-amplified DNA, which eliminated all related biases and inaccuracies.  the single molecule real time (SMRT) platform pioneerd by the Pacific biosciences company is the most utilized third-generation technology to date . The Pac bio technology have many beneficial features not shared by most other commercial technologies. For instance, Pac bio machinery generate extremely long reads exceeding 10kb in magnitude, which is convenient for de novo genome manufacturing.  Currently, the most anticipated field of third/fourth generation DNA sequencining technologies, is the assurance of nanopore sequencing, useful for identification of chemical and biological molecules .85. Interestingly the possibility of nanopore sequencing was set in stone even during first generation sequencing. As researchers showed that by electrophoresis, single DNA or RNA strands could be pumped across lipid bilayers via a-hemolysin ion channels. Furthermore, entry via the channel restricts the flow of ions, which reducing the current for a period of time proportional to the magnitude of the nucleic acid. The most recent advancement  Conclusion  Over the last 50years researchers worldwide have invented time and resources refining DNA sequencing technologies. At the start of this field, research was predominately done on accessible RNA targets.Next, revelations in sequencing protocols along with molecular biological activity enabled DNA readings to span over 200 hundred base pairs long, and sequencing processes to become parallel. .