The minimal catalytic mechanism of single-nucleotide incorporation by DNA pol has been proposed Donlin et al. A brief description for each reaction step can be found in the figure legend. In this aspect, none of the X and Y-family pols can meet this requirement. Both X and Y-family Pols have much lower nucleotide incorporation efficiency Brown et al. Therefore, they are not ideal for DNA sequencing. These repair pols generally make errors during DNA synthesis Kunkel and Bebenek, ; Kunkel, , and are not appropriate for high-precision DNA sequencing applications.
Under these parameters, the enzyme remains associated with the template DNA, it carries out sequential rounds of nucleotide incorporation until it dissociates from the binary complex Figure 1 , steps 2—7. In contrast, most cellular DNA replicases from A, B, C, and D families are distributive, and limited to only a few nucleotide incorporations. No X, Y, or RT-family enzymes are processive. Components of these replicative holoezymes are difficult to purify, and whole enzyme complexes are very challenging to reconstitute. Therefore, these types of enzyme complexes are seldom used in any DNA sequencing chemistry.
The minimal catalytic steps required for single-nucleotide incorporation by DNA polymerase. The k d, dNTP denotes the nucleotide binding constant of the enzyme. The actual chemistry happens step 5. This is followed by a second conformational change of the enzyme step 6 , which allows the final release of the PP i leaving group step 7. The nucleotide incorporation cycle is complete after PPi release. If the enzyme remains associated with DNA, a new round of nucleotide addition will continue until the enzyme dissociates from the DNA processive synthesis.
Generations of DNA polymerase-based sequencing methods and their corresponding commercial platforms are summarized in Table 2. As shown in Table 2 , all methods require a DNA polymerase to catalyze the necessary biochemical reaction for extracting DNA sequence information. The fundamental difference amongst these technologies is the type of nucleotide substrate incorporated. The structures of these nucleotides are illustrated in Figure 2. More in-depth information regarding these nucleotides can be found in the following articles Metzker et al.
From classical Sanger sequencing to modern NGS technologies, the nucleotide substrates used for sequencing have changed over time. The resulting dideoxy-terminated DNA fragments must be analyzed side-by-side using slab gel electrophoresis while sequence information is deduced via autoradiography Sanger et al.
The procedure itself is extremely time consuming and further compounded by low data output. This makes such an approach insufficient at meeting the growing demand for high-throughput DNA sequencing. Structures of nucleotides utilized in the generations of DNA polymerase-based sequencing methods. As a result, the misincorporated nucleotide is removed and the enzyme is ready to incorporate the correct nucleotide see Panel B , left to right cartoons. In the panels B,C , each filled circle indicates a nucleotide base.
A string of filled-gray circles represents the primer strand, and a string of filled-blue circles is the template DNA strand. Specific bases dC, dG, and dT are indicated inside the circles.
The advantages to this approach are 1 the four reaction mixtures can be combined and analyzed in a single sequencing lane; 2 the results can be directly monitored by a computer-aided fluorescence detection system, specifically matched to the emission spectra of the four dyes. These advantages allow DNA sequence information to be analyzed automatically by the computer. To solve this problem, the fluorescently labeled chain-terminating ddNTPs dye-terminators were soon introduced by Prober et al. Similar to the dye-primers, a set of fluorescently distinguished fluorophores are covalently attached to each of four ddNTPs See Figure 2C.
Adaptation of dye-terminators for Sanger sequencing workflow makes the four, base-specific chain termination reactions happen in one, single reaction tube. DNA polymerase is able to simultaneously incorporate four dye-terminators and generate the terminated DNA pieces for sequence analysis Rosenthal and Charnock-Jones, , The speed and throughput of dye-terminator sequencing was drastically improved when the automated capillary-array electrophoresis CAE was adopted for DNA analysis Drossman et al.
The dye-terminator-CE method has greatly improved sequencing performance and has become the laboratory standard for DNA sequencing over the past few decades. However, the technique itself is still very limited, especially for large-scale, whole genome sequencing. Increasing the sequencing throughput of dye-terminator-CE chemistry requires additional capillary tubes to be implemented. This becomes impractical for the application of high-throughput, multiplexing sequencing that is capable of sequencing millions of different DNA strands concurrently.
To alleviate this limitation, reversible dye-terminators were introduced to the modified, dye-terminating sequencing scheme. When these reversible dye-terminators are used in parallel with immobilization of DNA molecules on a solid-state surface, the individual DNA sequence can be directly ascertained from the base-specific, terminated DNA molecules recognized by the fluorescent imaging system Bentley et al.
As a result, the requirements for capillary electrophoresis CE analysis in a typical dye-terminator approach are no longer necessary, and millions of different DNA molecules can be sequenced simultaneously.
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DNA chains can thus be further extended by the DNA polymerase and incorporation can resume once more in the next reaction cycle Bentley et al. In both classes of reversible dye-terminators, cleavage of the linker group carrying the fluorescent dye leaves extra chemical molecules on the normal purine and pyrimidine bases. These molecular remnants may perturb the protein—DNA interaction and eventually impact the sequencing performance of the DNA polymerase Metzker, ; Chen et al. Hence, any fluorophore covalently attached to the PPi leaving group will be released after nucleotide addition to the primer terminus, and thus leave no molecular vestige in the DNA.
Since the added nucleotide possesses no blockage group to hinder DNA elongation from the primer terminus, the sequencing reaction can continue uninterrupted. Finally, there are no DNA scar issues for both pyrosequencing technology Ronaghi et al. Both technologies utilize natural nucleoside triphosphates dNTPs for their sequencing reactions Table 2 and Figure 2A.
A series of nucleotide modifications, created for rapidly changing DNA polymerase-based sequencing technologies has created a daunting task for DNA polymerase researchers to look for, design or evolve compatible enzymes for ever-changing DNA sequencing chemistries. From the beginning, A-family E. Sanger for his dideoxy-sequencing chemistry Sanger et al. However, Pol I effectively discriminates between a deoxy- and dideoxyribose in the nucleoside triphosphate, and does not incorporate ddNTPs very well Atkinson et al. This sequence-specific ddNTP incorporation by Pol I creates non-uniform band intensities on the sequencing gel.
Thus, the intensities of dideoxy-terminated bands are significantly more uniform with T7 pol in Sanger sequencing. To understand the molecular basis for this discrepancy, sequence analysis and biochemical studies were conducted among these three, A-family enzymes. Additionally, these two mutant proteins were demonstrated to incorporate fluorescein- and rhodamine-labeled dye-terminators, three orders of magnitude more efficiently than their wild-type parent enzymes Tabor and Richardson, However, Taq pol has become preferred for dye-terminator sequencing, because the enzyme has several advantages over Pol I or T7.
The enzyme is more readily purified and modifiable for further improvement. The Phe to Tyr mutation at position on conserved motif B of Taq pol only addresses the deoxy- and dideoxyribose selectivity problem in dye-terminator sequencing.
The enzyme, like Pol I, possesses bias. Kinetic analysis reveals that Taq pol favors ddGTP incorporation over other ddNTPs, with a much more robust nucleotide incorporation rate k pol ; Brandis et al. To investigate the cause of ddGTP bias, structural analysis of all four, ddNTP-trapped ternary complexes of the large fragment of Taq pol Klentaq1 was implemented. Substitution of the Arg residue with a negatively charged aspartic acid completely eliminates preference for ddGTP incorporation.
The enzyme variant bearing AL and YV double mutations on conserved motifs A and B, respectively, of the DNA polymerase shows enhanced preference for incorporating both acyclic and dideoxy dye-terminators over the parent enzyme Gardner and Jack, The same mutational effects were also found in enzyme mutants possessing homologous mutations in other archaeal, B-family DNA polymerase species Gardner and Jack, ; Gardner et al. JDF-3 also shows an additive effect on improving dye-terminator incorporation Arezi et al.
Using the structure-guided reconstruction of ancestral DNA sequence analysis on Taq pol, a library of 93 protein variants carrying different combinations of mutations were designed and screened for the ability to incorporate dNTP-ONH 2 in primer-extension assays.
DNA sequencing at past, present and future | Nature
One beneficial mutation LA on Taq pol was identified. The path toward acquisition of a compatible DNA polymerase for incorporation of fluorescent, terminal polyphosphate-labeled nucleotides has not been so straightforward. These techniques utilize DNA polymerase as a traditional incorporating enzyme, and alternatively as a molecular motor, responsible for controlled DNA translocation across the protein nanopore. In this approach, the enzyme functions as both DNA replicative enzyme, and molecular motor, which control the speed of DNA translocation through the MspA nanopore.
Unfortunately, this study is currently called into question, and the merits of this particular method must be reevaluated Chen et al. The large-scale of organism-specific, genome research reveals the intrinsic diversity and unique characteristics of DNA polymerases present in all kingdoms of life, including their viruses. Diverse DNA polymerases with distinct functions and properties provide a large pool of natural protein variants that can be tested, and later utilized, for continuously evolving sequencing-chemistries.
Tailor-made protein variants designed via protein engineering or directed-enzyme evolution have created powerful protein-engines that have propelled the progression of DNA sequencing technologies over the past few decades. Without a doubt, DNA polymerase has been, and will continue to remain, a crucial component of future sequencing technologies. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author thanks Ali Nikoomanzar for editing the manuscript, and Dr.
Lawrence A. Loeb, Eddie Fox, and Thomas Lie for critical reading the manuscript. Alexandrova, L. Nucleic Acids Res. Arezi, B. Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant. Arzumanov, A. Atkinson, M. Enzymatic synthesis of deoxyribonucleic acid. Biochemistry 8, — Bentley, D.
Accurate whole human genome sequencing using reversible terminator chemistry. Nature , 53— Blackburn, E.
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Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging. Blanco, L. Bloom, L. The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies. Brandis, J. Biochemistry 35, — Our conversation begins with a higher-level survey of the field -- one which cleanly and clearly defines CRISPR by placing it into a broader, and also a quite fascinating framework.
We cover four topics, which I'll now define up-front for you, so as to make the interview more accessible. We begin by discussing genetic sequencing. It's written in a limited alphabet, of just four letters: A, G, C, and T. And if someone sequences your genome, it simply means they've read it. They haven't modified it in any way.
They haven't have cloned you. They've just gotten a readout kind of like determining your blood type -- only a few billions times more complicated. George and I next discuss gene editing. As the word suggests, editing the genome of a person, bacterium, or virus involves changing some of its letters. This can significantly change an organism's function -- perhaps causing a small critter to produce something useful, like a medicine, or a biofuel.
Applied Molecular Cloning: Present and Future for Aquaculture
Or, perhaps someday giving people or animals superpowers. But it's not the first one. It's the tenth one -- in a lineage that goes back decades. But it's not better than all techniques at all things. And it's definitely not the last form of editing.
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There's massive headroom for improvement in genetic editing, and CRISPR will be superseded many times by more powerful approaches in the future. The third thing we discuss is DNA synthesis. The chain is now ready for another round of addition. The idea is counter-intuitive because scientists often keep the enzyme around. But TdT is cheap and easy to make using bacteria, explained Dr. Jay Keasling, who directed the study.
The speed was astonishing: they could add a new DNA letter in just 10 to 20 seconds—roughly 18 times faster than current approaches. Although the snipping step still takes about a minute, which adds up when synthesizing long DNA strands, the new technique comes out on top compared to conventional methods in terms of speediness. Rather than weeks, making a new gene from scratch will likely only take a day.
Part of it is accuracy. So far, after decades of fine-tuning, conventional DNA synthesis hits a The dream of storing massive amounts of data in DNA could also be possible. This study is just a glimpse of the exponential future of synthetic biology.
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