-
[show abstract]
[hide abstract]
ABSTRACT: Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ∼1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.
Proceedings of the National Academy of Sciences 08/2012; 109(36):14508-13. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The somatic mutation theory of aging posits that the accumulation of mutations in the genetic material of somatic cells as a function of time results in a decrease in cellular function. In particular, the accumulation of random mutations may inactivate genes that are important for the functioning of the somatic cells of various organ systems of the adult, result in a decrease in organ function. When the organ function decreases below a critical level, death occurs. A significant amount of research has shown that somatic mutations play an important role in aging and a number of age related pathologies. In this review, we explore evidence for increases in somatic nuclear mutation burden with age and the consequences for aging, cancer, and neurodegeneration. We then review evidence for increases in mitochondrial mutation burden and the consequences for dysfunction in the disease processes.
Mechanisms of ageing and development 11/2011; 133(4):118-26. · 4.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼35-kb circular DNA genome of unusually high A/T content (>86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3'→5' exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T→C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.
Journal of Molecular Biology 07/2011; 410(1):27-38. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.
Proceedings of the National Academy of Sciences 03/2011; 108(15):6079-84. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The central role of RNA polymerase (RNAP) is to catalyze the processive synthesis of a growing RNA transcript. Recent structural and biophysical data have led to a deeper understanding of the nucleotide addition cycle and insight into the structure-function relationships that govern transcription elongation. In this review, we discuss kinetic data on nucleotide incorporation in the context of crystal structures, which show RNAP in multiple conformations. We present a facilitated Brownian ratchet model of nucleotide incorporation, in which templated NTP binding to a non-catalytic site in the main channel promotes the conformational changes that lead to opening of the catalytic site and translocation.
Current Opinion in Structural Biology 11/2009; 19(6):708-14. · 9.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We deposited graded-index SiO2 films using glancing angle deposition to produce high-transmission antireflection coatings on glass. Because of the accurate control over the thin-film microstructure provided by this technique, we were able to create graded densities with a Gaussian profile resulting in transmission values greater than 99.9% for a single-layer interface with bandwidths up to 460 nm. The graded-index layer also provides low reflectance at nonnormal angles of incidence with transmission values degrading little for incidence angles up to 30 degrees.
Applied Optics 09/2003; 42(22):4573-9. · 1.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We present a simple, versatile technique for the fabrication of large, three-dimensional gap photonic crystals using glancing angle deposition (GLAD). A tetragonal lattice suitable for a large photonic band gap (PBG) can be synthesized by a regular array of square spiral structures grown from a simple, prepatterned substrate using physical vapor deposition and advanced substrate motion. Tetragonal square spiral crystals with a predicted PBG of up to 15% for a silicon structure can be produced in the visible, NIR, and IR spectrum. These PBG's exhibit good stability for large areas of parameter space that can be readily mapped out by the GLAD process through the variation of deposition parameters. Photonic crystals are optical materials renowned for their ability to restrict the propagation of certain wavelengths of light that fall within the "photonic band gap" (PBG) of the structure.
02/2003;
-
[show abstract]
[hide abstract]
ABSTRACT: We present a simple, versatile technique for the fabrication of large, three-dimensional gap photonic crystals using glancing angle deposition (GLAD). A tetragonal lattice suitable for a large photonic band gap (PBG) can be synthesized by a regular array of square spiral structures grown from a simple, prepatterned substrate using physical vapor deposition and advanced substrate motion. Tetragonal square spiral crystals with a predicted PBG of up to 15% for a silicon structure can be produced in the visible, NIR, and IR spectrum. These PBG's exhibit good stability for large areas of parameter space that can be readily mapped out by the GLAD process through the variation of deposition parameters.
11/2001;
-
[show abstract]
[hide abstract]
ABSTRACT: We report the fabrication and optical characterization of a tetragonal square spiral photonic crystal with a three-dimensional relative band gap of approximately 10% using the glancing angle deposition (GLAD) technique. This thin film structure is produced in a one-step process that is highly versatile as a wide range of crystal structures can be created simply through the variation of deposition parameters. Measurements indicate upper and lower frequency band edges at vacuum wavelengths of 2.50 and 2.75 μm, in the infrared region of the spectrum.
Photonics and Nanostructures - Fundamentals and Applications.
-
[show abstract]
[hide abstract]
ABSTRACT: We present a simple, versatile technique for the fabrication of large, three-dimensional gap photonic crystals using glancing angle deposition (GLAD). A tetragonal lattice suitable for a large photonic band gap (PBG) can be synthesized by a regular array of square spiral structures grown from a simple, prepatterned substrate using physical vapor deposition and advanced substrate motion. Tetragonal square spiral crystals with a predicted PBG of up to 15% for a silicon structure can be produced in the visible, NIR, and IR spectrum. These PBG's exhibit good stability for large areas of parameter space that can be readily mapped out by the GLAD process through the variation of deposition parameters. Photonic crystals are optical materials renowned for their ability to restrict the propagation of certain wavelengths of light that fall within the "photonic band gap" (PBG) of the structure. 1,2 PBG materials were first proposed in the context of light localization 1 and inhibited spontaneous emission, 2 where applications include high-efficiency lasers, all-optical microtransistors and optical microcircuitry. 3 The common difficulty with three-dimensional band gap structures, how-ever, is that fabrication is extremely difficult and traditionally involves numerous microelectronic and/or lithographic pro-cessing steps and techniques. We present a technique that circumvents these complications, in which fabrication of photonic crystals is virtually reduced to a single-step process and results in an optimal crystal structure. The diamond lattice structure has traditionally been considered an ideal candidate for a large PBG opening between the second and third photon dispersion bands (fundamental gap). 4 However, its complexity has meant that traditional techniques make successful production of such a crystal even more difficult than for those of the more standard and already complex bcc and fcc lattices. This has motivated efforts to mimic the diamond lattice using woodpile struc-tures 5 and criss-crossing pore structures, 6 which are neverthe-less problematic to fabricate on a large scale. Observation of the helical atomic arrangement in the diamond unit cell motivated Chutinan and Noda to consider circular spiral post structures, 7 yet these circular spiral posts exhibit a phase shift between adjacent posts, rendering them impractical for microfabrication.
