Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation

Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]     

Rearrangement of the (6S,8R,11S) and (6R,8S,11R) exocyclic 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal to N2-deoxyguanosine cyclic hemiacetal adducts when placed complementary to cytosine in duplex DNA

trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. The Michael addition of deoxyguanosine to HNE yields four diastereomeric exocyclic 1,N(2)-dG adducts. The corresponding acrolein- and crotonaldehyde-derived exocyclic 1,N(2)-dG adducts undergo ring-opening to N(2)-dG aldehydes, placing the aldehyde functionalities into the minor groove of DNA. The acrolein- and the 6R-crotonaldehyde-derived exocyclic 1,N(2)-dG adducts form interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Only the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Moreover, as compared to the exocyclic 1,N(2)-dG adducts of acrolein and crotonaldehyde, the cross-linking reaction is slow (Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc. 2003, 125, 5687-5700). Accordingly, the chemistry of the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry has been compared with that of the (6R,8S,11R) adduct, when incorporated into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3', containing the 5'-CpG-3' sequence (X = HNE-dG). When placed complementary to dC in this duplex, both adducts open to the corresponding N(2)-dG aldehydic rearrangement products, suggesting that the formation of the interstrand cross-link by the exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry, and the lack of cross-link formation by the exocyclic 1,N(2)-dG adduct of (6R,8S,11R) stereochemistry, is not attributable to inability to undergo ring-opening to the aldehydes in duplex DNA. Instead, these aldehydic rearrangement products exist in equilibrium with stereoisomeric cyclic hemiacetals. The latter are the predominant species present at equilibrium. The trans configuration of the HNE H6 and H8 protons is preferred. The presence of these cyclic hemiacetals in duplex DNA is significant as they mask the aldehyde species necessary for interstrand cross-link formation.     

High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry

An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2′-deoxycytidine (5MedC) to 2′-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC–ESI-MS/MS. The cost-effective internal standards ¹⁵N₃-dC and ¹⁵N₅-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4 femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring.     

Interstrand cross-link formation in duplex and triplex DNA by modified pyrimidines

DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.     

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 +/- 1.2 8-oxodG per 10(6) unmodified nucleotides (n = 5) using 5 microg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5.     

苯并芘DNA加合物anti-BPDE-N^2-dG四种立体异构体的合成及色谱分离

体外合成了苯并芘DNA加合物-邻二醇环氧苯并芘-脱氧鸟苷加合物(anti-BPDE-N2-dG)四种立体异构体(两对手性异构体)。通过优化体外反应条件,anti-BPDE-N2-dG四种异构体的合成产量较现有合成方法提高了2倍多,为定量检测生物体中anti-BPDE-N2-dG提供了标准品。并首次将五氟苯基色谱柱应用于该立体异构体的色谱分离提纯,通过优化色谱条件,采用常规的五氟苯基色谱柱(250 mm×4.6 mm,5μm),以乙腈-0.1%甲酸水(22.5∶77.5)为流动相,流速1.2 mL/min条件下,45 min内即可分离提纯四种立体异构体。该方法与常规C18柱(250 mm×4.6 mm,5μm)需要160 min,苯基柱(250 mm×4.6 mm,5μm)需要85~100 min才能将四种立体异构体实现色谱分离相比,缩短了分离时间,提高了提纯效率。通过紫外吸收光谱、质谱、圆二色谱对四种立体异构体进行表征,确定出峰顺序为trans(-)、trans(+)、cis(+)、cis(-)-anti-BPDE-N2-dG。此外,利用高效液相色谱-串联质谱(HPLC-MS/MS)检测anti-BPDE-N2-dG四种立体异构体标准品时,使用常规的五氟苯基色谱柱可在30 min内完成分离检测,与相同规格的苯基柱需要60 min相比提高了检测效率。     

Formation of a N2-dG:N2-dG carbinolamine DNA cross-link by the trans-4-hydroxynonenal-derived (6S,8R,11S) 1,N2-dG adduct

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.     

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

Bypass of aflatoxin B1 adducts by the Sulfolobus solfataricus DNA polymerase IV

Aflatoxin B(1) (AFB(1)) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB(1)-N7-dG). The AFB(1)-N7-dG can rearrange to a formamidopyrimidine (AFB(1)-FAPY) derivative. Both AFB(1)-N7-dG and the β-anomer of the AFB(1)-FAPY adduct yield G→T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB(1)-N7-dG in an error-free manner but conducts error-prone replication past the AFB(1)-FAPY adduct, including misinsertion of dATP, consistent with the G→T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB(1)-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB(1)-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB(1)-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O(2) oxygen of dTTP, and between the template T O(4) oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB(1)-N7-dG maintains the 5'-intercalation of the AFB(1) moiety observed in DNA. The bond between N7-dG and C8 of the AFB(1) moiety remains in plane with the alkylated guanine, creating a 16° inclination of the AFB(1) moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB(1)-FAPY adducted template:primer also maintains 5'-intercalation of the AFB(1) moiety. The β-deoxyribose anomer is observed. Rotation about the FAPY C5-N(5) bond orients the bond between N(5) and C8 of the AFB(1) moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves stacking of the AFB(1) moiety above the 5'-face of the FAPY base, as compared to the AFB(1)-N7-dG adduct. Ternary structures with AFB(1)-β-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB(1)-β-FAPY:dC pair. For dATP, the oxygen atom of the FAPY formamide group participates in a water-mediated hydrogen bond with Arg332. The insertion of dTTP yields a structure similar to that observed for the AFB(1)-N7-dG adduct. The differential accommodation of these AFB(1) adducts within the active site may, in part, modulate lesion bypass.     

戊二醛合成跟踪分析

用气相色谱法对液 液相转移反应萃取法合成戊二醛（ＧＡ）反应进行跟踪分析,采用φ３ｍｍ×４ｍ［ｍ（ＳＥ ３０）∶ｍ（硅烷化１０１白色担体）＝１５∶１００］＋φ３ｍｍ×２ｍ［ｍ（ＰＥＧ ２０Ｍ）∶ｍ（硅烷化１０１白色担体）＝１０∶１００］不锈钢柱,双柱双氢焰程序升温,升温程序为７０℃（４ｍｉｎ）１０℃／ｍｉｎ１４０℃（６ｍｉｎ）。Ｎ２４５ｍＬ／ｍｉｎ,Ｈ２６５ｍＬ／ｍｉｎ,空气３００ｍＬ／ｍｉｎ,汽化温度１８５℃,检测温度１８５℃。所建立的方法能较好地分离环戊烯、溶剂、氧化中间产物、ＧＡ、内标、氧化副产物。     

Anomeric and Enantiomeric 2’-Deoxycytidines: Base Pair Stability in the Absence and Presence of Silver Ions

Dodecamer duplex DNA containing anomeric (α/β-d ) and enantiomeric (β-l /β-d ) 2’-deoxycytidine mismatches was studied with respect to base pair stability in the absence and presence of silver ions. Stable duplexes with silver-mediated cytosine–cytosine pairs were formed by all anomeric and enantiomeric combinations. Stability changes were observed depending on the composition of the mismatches. Most strikingly, the new silver-mediated base pair of anomeric α-d -dC with enantiomeric β-l -dC is superior to the well-noted β-d /β-d -dC pair in terms of stability. CD spectra were used to follow global helical changes of DNA structure.     

Thermodynamic versus kinetic products of DNA alkylation as modeled by reaction of deoxyadenosine.

Alkylating agents that react through highly electrophilic quinone methide intermediates often express a specificity for the weakly nucleophilic exocyclic amines of deoxyguanosine (dG N(2)) and deoxyadenosine (dA N(6)) in DNA. Investigations now indicate that the most nucleophilic site of dA (N1) preferentially, but reversibly, conjugates to a model ortho-quinone methide. Ultimately, the thermodynamically stable dA N(6) isomer accumulates by trapping the quinone methide that is transiently regenerated from collapse of the dA N1 adduct. Alternative conversions of the dA N1 to the dA N(6) derivative by a Dimroth rearrangement or other intramolecular processes are not competitive under neutral conditions, as demonstrated by studies with [6-(15)N]-dA. Both a model quinone methide precursor and its dA N1 adduct yield a similar profile of deoxynucleoside products when treated with an equimolar mixture of dC, dA, dG, and T. Consequently, the most readily observed products of DNA modification resulting from reversible reactions may reflect thermodynamic rather than kinetic selectivity.     

Functionalized Silver-Ion-Mediated α-dC/β-dC Hybrid Base Pairs with Exceptional Stability: α-d-5-Iodo-2′-Deoxycytidine and Its Octadiynyl Derivative in Metal DNA

Silver-mediated α-dC–Ag⁺–β-dC hybrid base pairs decorated with 5-iodo- or 5-octadiynyl residues are well accommodated in duplex DNA. A strong T_m increase and favorable thermodynamic data for duplex DNA were observed after addition of silver ions. The phenomenon is particularly obvious when both nucleobases of the base pairs are functionalized. Neither the position of the base pair, nor the type of 5-substituent had a negative influence. On the contrary, functionalization of conventional silver-mediated β-dC–Ag⁺–β-dC homo base pairs showed a negative impact induced by the bulky substituents. To this end, cytosine modified 12-mer oligodeoxynucleotides were prepared by solid-phase synthesis employing new α-anomeric 2′-deoxycytidine phosphoramidites. A multigram scale synthesis was developed for 5-iodo-α-d -2′-deoxycytidine ( 1 ) employing the direct glycosylation of cytosine with Hoffer's α-d -halogenose followed by separation of anomeric DMT nucleosides. Regarding base-pair stability and functionalization silver-mediated α/β-dC hybrid base pairs were found to be superior to β/β-dC homo pairs. According to their extraordinary properties, they might find applications in DNA diagnostics, material science, or nanotechnology.     

MODIFICATION OF MEMBRANE COMPOSITION, EICOSANOID METABOLISM, AND IMMUNORESPONSIVENESS BY DIETARY OMEGA-3 AND OMEGA-6 FATTY ACID SOURCES, MODULATORS OF ULTRAVIOLET-CARCINOGENESIS

Dietary sources of lipids containing predominantly n-3 or n-6 fatty acids (FA) have been examined for effect upon several potential pathophysiologic parameters. Epidermal, plasma, and red blood cell (RBC) membrane FA composition exhibited marked differences between animals fed the respective dietary lipid sources. Reduced levels of 18:1, 20:3 and 20:4 occurred in the n-3 FA fed animals which exhibited significantly higher levels of 20:5 and 22:6. Approximately equal levels of 18:2 were present in animals fed either diet. Despite marked differences in RBC membrane FA composition, only marginal effect upon osmotic fragility occurred. Lower levels of 20:3 and 20:4 found in n-3 fed animals could result from a deficit of elongase and/or delta 5-desaturase activity. Whether lower 20:4 levels in n-3 fed animals could rate-limit eicosanoid metabolism is unknown, but epidermal capacity to metabolise arachidonic acid in these animals was found to be closely related to n-6 FA intake. Animals fed n-3 FA exhibited markedly lower levels of plasma PGE2, even when the diet was supplemented with n-6 FA. In addition, UV-radiated animals receiving the n-3 FA source demonstrated a reduced (approximately 30%) response to inflammatory stimulus and a greater (4.5-fold) delayed hypersensitivity (DH) to dinitrochlorobenzene than animals fed the n-6 FA source. These data demonstrate that dietary lipid strongly influences tissue FA composition, eicosanoid metabolism, and, in the case of DH, at least one type of T-cell mediated immune response in UV-irradiated animals.     

Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption

The current paper describes the design, fabrication, and testing of a micromachined submicroliter-volume polymerase chain reaction (PCR) chip with a fast thermal response and very low power consumption. The chip consists of a bulk-micromachined Si component and hot-embossed poly(methyl methacrylate)(PMMA) component. The Si component contains an integral microheater and temperature sensor on a thermally well-isolated membrane, while the PMMA component contains a submicroliter-volume PCR chamber, valves, and channels. The micro hot membrane under the submicroliter-volume chamber is a silicon oxide/silicon nitride/silicon oxide (O/N/O) diaphragm with a thickness of 1.9 microm, resulting in a very low thermal mass. In experiments, the proposed chip only required 45 mW to heat the reaction chamber to 92 degrees C, the denaturation temperature of DNA, plus the heating and cooling rates are about 80 degrees C s(-1) and 60 degrees C s(-1), respectively. We validated, from the fluorescence results from DNA stained with SYBR Green I, that the proposed chip amplified the DNA from vector clone, containing tumor suppressor gene BRCA 1 (127 base pairs at 11th exon), after 30 thermal cycles of 3 s, 5 s, and 5 s at 92 degrees C, 55 degrees C, and 72 degrees C, respectively, in a 200 nL-volume chamber. As for specificity of DNA products, owing to difficulty in analyzing the very small volume PCR results from the micro chip, we vicariously employed the larger volume PCR products after cycling with the same sustaining temperatures as with the micro chip but with much slower ramping rates (3.3 degrees C s(-1) when rising, 2.5 degrees C s(-1) when cooling) within circa 20 minutes on a commercial PCR machine and confirmed the specificity to BRCA 1 (127 base pairs) with agarose gel electrophoresis. Accordingly, the fabricated micro chip demonstrated a very low power consumption and rapid thermal response, both of which are crucial to the development of a fully integrated and battery-powered instrument for a lab-on-a-chip DNA analysis.     

Characterization of the cation and temperature dependence of DNA quadruplex hydrogen bond properties using high-resolution NMR

Variations in the hydrogen bond network of the Oxy-1.5 DNA guanine quadruplex have been monitored by trans-H-bond scalar couplings, (h2)J(N2N7), for Na(+)-, K(+)-, and NH(4)(+)-bound forms over a temperature range from 5 to 55 degrees C. The variations in (h2)J(N2N7) couplings exhibit an overall trend of Na(+) > K(+) > NH(4)(+) and correlate with the different cation positions and N2-H2...N7 H-bond lengths in the respective structures. A global weakening of the (h2)J(N2N7) couplings with increasing temperature for the three DNA quadruplex species is accompanied by a global increase of the acceptor (15)N7 chemical shifts. Above 35 degrees C, spectral heterogeneity indicates thermal denaturation for the Na(+)-bound form, whereas spectral homogeneity persists up to 55 degrees C for the K(+)- and NH(4)(+)-coordinated forms. The average relative change of the (h2)J(N2N7) couplings amounts to approximately 0.8 x 10(-3)/K and is thus considerably smaller than respective values reported for nucleic acid duplexes. The significantly higher thermal stability of H-bond geometries in the DNA quadruplexes can be rationalized by their cation coordination of the G-quartets and the extensive H-bond network between the four strands. A detailed analysis of individual (h2)J(N2N7) couplings reveals that the 5' strand end, comprising base pairs G1-G9* and G4*-G1, is the most thermolabile region of the DNA quadruplex in all three cation-bound forms.     

高效凝胶渗透色谱法检测马初乳中的免疫球蛋白G

建立了高效凝胶渗透色谱(HPGPC)测定马初乳中免疫球蛋白G(IgG)含量的方法。采用TOSOH TSK-G4000PWXL色谱柱(300 mm×7.8 mm, 5 μm)分离,以0.05 mol/L磷酸盐缓冲液(pH 6.9)为流动相,流速0.8 mL/min,检测波长280 nm,温度25 ℃。结果表明: 免疫球蛋白G的线性范围为0.2～3.0 g/L(r2=0.9995),平均回收率为97.47%,相对标准偏差(RSD)为1.22%,检出限(信噪比为10)为0.08 mg/L,方法的稳定性、精密度和重现性(以峰面积的RSD计)分别为2.86%、1.62%、1.82%。在优先满足小马哺育的前提下,采集新疆昭苏马场中两个不同品种马匹的马乳,于低温保存,在4 ℃和12000 r/min下30 min内离心两次,制得乳清,测得第一次泌乳时,IgG含量在2 h时高达35.0～50.0 g/L,而在72 h后,马乳中IgG含量迅速下降为2.0～4.0 g/L。该方法前处理过程简单、快速,方法简便、准确、重现性好、精密度高,适合作为马初乳中IgG的检测方法。     

Analysis of amino acids without derivatization in barley extracts by LC-MS-MS

A method has been developed for quantification of 20 amino acids as well as 13 (15)N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5micro SCX 100A) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d (2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.     

DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80 degrees C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N-dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDEA30). DNA-polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70 degrees -75 degrees C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50 degrees C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60 degrees C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50 degrees C. Molecular masses of these polymers were determined by tandem gel permeation chromatography-multiangle laser light scattering method (GPC-MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.     

Determination of vitamin E acid succinate in biodegradable microspheres by reversed-phase high-performance liquid chromatography

A simple, rapid, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method is applied to the routine assay of vitamin E acid succinate in biodegradable microspheres. Vitamin E acid-succinate-containing poly-(D,L-lactic-co-glycolic acid) microspheres are prepared by the solvent evaporation method. The starting drug-polymer ratio is 1:10 (w/w) and the total amount of drug and polymer processed is always 440 mg. The content of vitamin E acid succinate in the microspheres is evaluated by HPLC. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Extrasil ODS-2 column with a mobile phase composed of methanol-water (97:3, v/v) (pH 5.6) at a flow rate of 2 mL/min and UV detection at 284 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The stability of vitamin E acid succinate is also studied with satisfactory results after 48 h at 25 degrees C. The method is selective and linear for drug concentrations in the range 15-210 micro g/mL. The LOQ and LOD are 15 and 3 micro g/mL, respectively. The results for accuracy studies are good. Values for coefficient of variation for intra- and interassay are 2.08% and 2.32%, respectively. The mean percentage of vitamin E acid succinate in the recovery studies is 99.52% +/- 0.81%. The mean loading efficiency for microspheres is 96.53% +/- 1.31%.