Retroactive DNA analysis for sex determination and dystrophin gene by polymerase chain reaction with archived cytogenetic slides

We describe a rapid and efficient diagnostic method for sex determination and the dystrophin gene by the polymerase chain reaction (PCR) using archived cytogenetic slides. Archived cytogenetic slides stored for about 4 years at room temperature were used. To confirm whether DNA analysis is possible using the archived cytogenetic slides, we extracted the DNA from the slides and amplified the Y centromeric region (DYZ3), the X centromeric region (DXZ1) and the exon 46 of the dystrophin gene. Of the 50 cases, 24 were peripheral bloods, 13 were amniotic fluid cells, 5 were chorionic villus samplings and 8 were cord bloods. The PCR related sex determination in 22 females and 28 males, showed 100% concordance with the results of chromosome analysis, and all cases showed positive band for the exon 46 of the dystrophin gene. Of the 50 cases of the archived cytogenetic slides, we were fortunate enough to obtain the fresh blood sample from one fetus whose karyotype showed 45,X[34]/46,X,+mar[145] to compare the results of the gDNA with that from archived cytogenetic slide. To confirm whether the marker chromosome was derived from Y chromosome, we studied the six loci (PABY, SRY, RPS4Y (SY16, 17), ZFY, DYS14) on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm. Of the 8 loci studies, all PCR related Y chromosome showed positive band from both gDNA obtained from cord blood and archived cytogenetic slides. We could conclude from the above results that the marker chromosome was derived from the Y chromosome. We believe our experiment is rapid and efficient for studies of over 10 independent loci from a single slide which has been kept in storage for up to 4 years and that archival Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for clinical and forensic studies.     

The intramolecular mechanism for the second proton transfer in triosephosphate isomerase (TIM): a QM/FE approach

The intramolecular mechanism we earlier proposed [Alagona, G.; Desmeules, P.; Ghio, C.; Kollman, P. A. J Am Chem Soc 1984, 106, 3623] for the second proton transfer of the reaction catalyzed by triosephosphate isomerase (TIM) is examined ab initio at the HF and MP2/6-31+G** levels in vacuo for two conformers of the enediolate phosphate (ENEP), with the ethereal oxygen of the phosphate group either syn (X), as in the crystal structure, or anti (Y) with respect to the enediolate carbonyl O. The barrier height for the intramolecular proton transfer occurring in enediolate is very sensitive to electron correlation corrections. The MP2 internal energy barrier is much lower than the HF one, while the free energy (FE) barrier is even more favorable, indicating that the enzyme presence is not requested to speed up that step. An investigation of the dynamical aspects of the mechanism, along the pathway from ENEP A (with H on O(1)) to TS and from TS to ENEP B (with H on O(2)), was, however, carried out in the presence of the enzyme field while using a neutral His-95 with its proton on Ndelta. To perform the FE simulations, it was necessary to parametrize in the AMBER force-field the ENEP A, TS and B species, whose partial charges have been determined with the RESP procedure, with the X and Y arrangements of the phosphate head. Actually, the FE/QM approach produced a low barrier and a substantial balance between A and B, especially at the MP2 level. The trajectories, analyzed paying a particular attention to the positions assumed by His-95 and by the other active site residues, put forward somewhat different H-bond patterns around the X or Y enediolate phosphate.     

Topological characteristics of electron density distribution in SSXY --> XSSY (X or Y = F, Cl, Br, I) isomerization reactions

The reactions of SSXY to XSSY (X or Y = F, Cl, Br, I) have been studied at B3LYP/6-311++G(2df) and MP2/6-311++G(2df) levels based on B3LYP/6-311++G(2df) optimized geometries. There are two pathways (X or Y atom transferring) for each reaction of SSXY to XSSY(X or Y = F, Cl, Br, I). The "Atoms in Molecules" theory has been applied to analyze the topological characteristics of electron density distribution along the reaction path. (a) The S-S-X or S-S-Y ring structure transition region (STR) is contributed to S(1)-X or S(1)-Y bond formation and S(2)-X or S(2)-Y bond annihilation. The STR and structure transition state (STS) are defined. (b) Comparing the two pathways of each SSXY --> XSSY (X, Y = F, Cl, Br, I) reaction, X or Y atom transferring, the broader the STR is, the later the STS appears, and the pathway is easier. (c) When X linked to the same S site of a three-member ring (S-S-F, S-S-Cl, S-S-Br, or S-S-I), the ring STR becomes broader and the STS appears later according to the sequence of X = F, Cl, Br, and I. And in these cases, for exothermic reactions, the higher the DeltaE(P-->TS1) is, the broader the STR is and the later the STS appears. (d) When the same Y atom linked to different three-member-ring S-S-X, the STR becomes broader and the STS appears later according to the sequence of X = F, Cl, Br, and I in the ring. And in these cases, for exothermic reactions, the lower the DeltaE(P-->TS2) is, the broader the STR is and the later the STS appears.     

Theoretical study of halophilic reactions of stable silylenes with chloro- and bromocarbons

A theoretical study of the mechanism of the reaction of stable silylenes with halocarbons has been carried out using the B3LYP density functional method. The main findings are as follows: (1) Lewis acid-base complexes formed between silylenes and halocarbons do not play a role in silylene insertion chemistry into halocarbons; therefore, the acid-base complex mechanism proposed by West et al. (J. Am. Chem. Soc. 2002, 124, 4186) is not appropriate to describe the disilane formation reaction. (2) The disilane formation reactions follow the energetically favorable general reaction pathway (X = halogen): (i) Y2Si: + HCX3 --> TS1 --> Y2XSi-CHX2. (ii) Y2Si: + Y2XSi-CHX2 --> TS2 --> Y2XSi-SiY2CHX2. (3) The observed preference of stable silylenes to undergo C-X bond insertion rather than C-H bond has been investigated. The theoretical findings suggest that this preference is a result of the thermodynamic factor. (4) Stable silylenes prefer to insert into a C-Br rather than a C-Cl bond because the energy barrier to insertion is lower, and the reaction is more exothermic.     

Combination of 768-well microplate array diagonal gel electrophoresis with duplex PCR of X and Y chromosome markers for quality control of epidemiological DNA banks

Large DNA banks for human epidemiological studies have become an increasingly important research tool. The power of genotype-phenotype studies is dependent both on the quality of phenotyping and of genotyping and of correct linking of phenotypes to genotypes. Samples must be tracked through numerous steps between subject or patient and post-genotypic data. Only one phenotype, sex, has a perfect and binary correlation with genotype. In mixed sex studies, it may be advantageous for purposes of quality control to keep sexes mixed during the steps from acquisition to DNA bank, in order to be able to check later for sample swaps. We have designed a duplex PCR combining an amplicon from MAOA marking the X chromosome and an amplicon from DDX3Y marking the Y chromosome. We combined this with a simple economical palmtop sized 768-well microplate compatible electrophoresis system developed in-house for examination of duplex PCR products. We applied this quality control test in the validation of two DNA banks.     

Ab initio molecular orbital studies of nonidentity allyl transfer reactions

Ab initio molecular orbital (MO) calculations are carried out on the nonidentity allyl transfer processes, X^? + CH₂CHCH₂Y ? CH₂CHCH₂ X + Y^?, with X^? = H, F, and Cl and Y = H, NH₂, OH, F, PH₂, SH, and Cl. The Marcus equation applies well to the allyl transfer reactions. The transition state (TS) position along the reaction coordinate and the TS structure are strongly influenced by the thermodynamic driving force, whereas the TS looseness is originated from the intrinsic barrier. The intrinsic barrier, ΔE, looseness, %L?, and absolute asymmetry, %AS?, are well correlated with the percentage bond elongation, %CY? = [(d ? d)/d] × 100 and/or %CX?. The %CY? and the bond orders indicate that a stronger nucleophile and/or a stronger nucleofuge (or a better leaving group) leads to an earlier TS on the reaction coordinate with a lesser degree of bond making as well as bond breaking. These are consistent with the Bell-Evans-Polanyi principle and the Leffler-Hammond postulate. © 1995 by John Wiley & Sons, Inc.     

An analysis of the interaction energy in some SN2 reactions

The interaction energy between an incoming group X^– and the substrate CRH₂Y at the geometry of the transition state (TS) for bimolecular nucleophilic substitution reactions (with X, Y, and R equal to H and F) has been subjected to decomposition according to the Morokuma scheme. The influence of the basis set and of the geometry chosen for the TS is examined. The results bring out regular trends in the different terms of the decomposition along the whole set of reactions, but they are not sufficient to give a rationale of the energetic factors involved in these reactions.     

Resonance‐Assisted Intramolecular Chalcogen–Chalcogen Interactions?

High-level B3LYP/6-311+G(3df,2p) density functional calculations have been carried out for a series of saturated chalcogenoaldehydes: CH(X)-CH(2)-CH(2)YH (X, Y=O, S, Se, Te). Our results indicate that in CH(X)-CH(2)-CH(2)YH (X=Y=O, S, Se) the X-H...X intramolecular hydrogen bond (IHB) competes in strength with the X...XH chalcogen-chalcogen interaction, while the opposite is found for the corresponding tellurium-containing analogues. For those derivatives in which X does not equal Y, X being the more electronegative atom, the situation is more complicated due to the existence of two non-equivalent X-H and Y-H tautomers. The Y-H tautomer is found to be lower in energy than the X-H tautomer, independently of the nature of X and Y. For X=O, S, Se and Y=S, Se the most stable conformer b is the one exhibiting a Y-H...X IHB. Conversely when Y=Te, the chelated conformer d, stabilized through a X...YH chalcogen-chalcogen interaction is the global minimum of the potential energy surface. Systematically the IHB and the chalcogen-chalcogen interactions observed for saturated compounds are much weaker than those found for their unsaturated analogues. This result implies that the nonbonding interactions involving chalcogen atoms, mainly Se and Te, are not always strongly stabilizing. This conclusion is in agreement with the fact that intermolecular interactions between Se and Te containing systems with bases bearing dative groups are very weak. We have also shown that these interactions are enhanced for unsaturated compounds, through an increase of the charge delocalization within the system, in a mechanism rather similar to the so call Resonance Assisted Hydrogen Bonds (RAHB). The chalcogen-chalcogen interactions will be also large, due to the enhancement of the X-->Y dative bond, if the molecular environment forces the interacting atoms X and Y to be close each other.     

A Study on Deletion in Polymer Region of Amelogenin (Amel Y) Gene: Significant Importance in Sex Determination and Identification of Sexual Assault Perpetrator

Accurate gender determination is crucial in many scientific disciplines, especially in prenatal diagnosis of X-linked diseases and forensic investigations. Today, molecular techniques, especially typing for a length variation in the X–Y homologous amelogenin gene (AMEL X and AMEL Y), are used for gender assignation. This amelogenin is an integral part of most PCR multiplex kits for gender determination marker, but in 1998 there was a report of two normal males being typed as female with this marker. Subsequently, a small number of males with amelogenin deleted genes have been reported in various populations but little data are available characterizing these deletions. This review aims to explore possible relationships among the AMEL Y deleted samples and Y-chromosome microsatellite haplotypes. Also, attempts are made to determine the frequency of males with AMEL Y deleted gene in various countries across the globe. Although some studies have shown that males with AMEL Y deleted gene are extremely rare in most populations, typing an additional gender-determining locus should be considered in forensic investigations where the reference sample is of unknown gender.     

Finding Y‐relevant part of X by use of PCR and PLSR model reduction methods

The paper is considering the following question: using principal component regression (PCR) or partial least squares regression (PLSR), how much data can be removed from X while retaining the original ability to predict Y ? Two model reduction methods using similarity transformations are discussed, one giving projections of original loadings onto the column space of the fitted response matrix (essentially the orthogonal signal correction (OSC) methods), and one giving projections of original scores onto the column space of the coefficient matrix (essentially the net analyte signal (NAS) methods). The loading projection method gives model residuals that are orthogonal to Y and , which is valuable in certain applications. The score projection method, on the other hand, gives model residuals that are orthogonal to , which is essential in other applications. It is shown that the reduced matrix X from the score projection method is a subset of the reduced matrix X from the loading projection method. It therefore has the smallest Frobenius norm, and thus the smallest total column variance, assuming centered data. Copyright © 2007 John Wiley & Sons, Ltd.     

Computational study on the reaction pathway of α-bromoacetophenones with hydroxide ion: possible path bifurcation in the addition/substitution mechanism

The reaction of an α-haloketone with a nucleophile has three reaction channels: carbonyl addition, direct substitution, and proton abstraction. DFT calculations for the reaction of PhCOCH(2)Br with OH(-) showed that there exists an addition/substitution TS on the potential energy surface, in which OH(-) interacts with both the α- and carbonyl carbons. The intrinsic reaction coordinate calculations revealed that the TS serves as the TS for direct substitution for XC(6)H(4)COCH(2)Br with an electron-donating X or a X less electron-withdrawing than m-Cl, whereas the TS serves as the TS for carbonyl addition for derivatives with a X more electron-withdrawing than m-CF(3). Trajectory calculations starting at respective TS indicated that the single TS can serve for the two mechanisms, substitution and addition, through path bifurcation after the TS for borderline substrates. The reaction is the first example of dynamic path bifurcation for fundamental reaction types of carbonyl addition and substitution.     

Origin of diastereoselectivity in the thermal [4+2] cycloadditions of dienophiles derived from Oppolzer's sultams: Steric vs. Stereoelectronic Influences

Comparative semi-empirical PM3 and ab initio STO 3-21G calculations on bornanesultam-derived dienophiles containing the structural moiety SO₂? N? C(O)? X(α) = Y(β) suggest that, among the conformers of low energy, the thermodynamically less stable SO₂/C(O)-syn,C(O)/X=Y-s-cis conformation is also reactive in terms of LUMO level and atomic coefficients. Furthermore, the X(α), Y(β) LUMO atomic coefficients are nonequivalent with respect to both X(α)-re and X(α)-si faces, and thus have, depending on the conformation, a matching or mismatching stereoelectronic influence with the co-operative steric effect. This dissymmetry is believed to result from the generalized anemone effect of the N lone pair, itself anomerically stabilized and directed, in the absence of crucial steric interactions, by the pseudo-axial anti-periplanar S?O bond. Five N-acyl-substituted bornanesultams arc discussed ((–)- 1a : N-acryloyl, X?CH, Y?CH₂; (–)- 1b ; N-crotonoyl, X?CH, Y?CHMe; (–)- 1c : N-N′-fumaroyl, X?CH, Y?CH(C(O)-bornanesultam); 2a : N-glyoxyloyl, X?CH, Y?O; 2b : N-acylnitroso, X?N, Y?O). In this context, differences with toluenesultams 3 are pointed out. A previous report on N-(acylnitroso)-bornanesultam 2b is revisited, and the diastereoselectivity observed is shown to result from thermodynamic control.     

Chemical dynamics simulations of X- + CH3Y → XCH3 + Y- gas-phase S(N)2 nucleophilic substitution reactions. Nonstatistical dynamics and nontraditional reaction mechanisms

Extensive classical chemical dynamics simulations of gas-phase X(-) + CH(3)Y → XCH(3) + Y(-) S(N)2 nucleophilic substitution reactions are reviewed and discussed and compared with experimental measurements and predictions of theoretical models. The primary emphasis is on reactions for which X and Y are halogen atoms. Both reactions with the traditional potential energy surface (PES), which include pre- and postreaction potential energy minima and a central barrier, and reactions with nontraditional PESs are considered. These S(N)2 reactions exhibit important nonstatistical atomic-level dynamics. The X(-) + CH(3)Y → X(-)---CH(3)Y association rate constant is less than the capture model as a result of inefficient energy transfer from X(-)+ CH(3)Y relative translation to CH(3)Y rotation and vibration. There is weak coupling between the low-frequency intermolecular modes of the X(-)---CH(3)Y complex and higher frequency CH(3)Y intramolecular modes, resulting in non-RRKM kinetics for X(-)---CH(3)Y unimolecular decomposition. Recrossings of the [X--CH(3)--Y](-) central barrier is important. As a result of the above dynamics, the relative translational energy and temperature dependencies of the S(N)2 rate constants are not accurately given by statistical theory. The nonstatistical dynamics results in nonstatistical partitioning of the available energy to XCH(3) +Y(-) reaction products. Besides the indirect, complex forming atomic-level mechanism for the S(N)2 reaction, direct mechanisms promoted by X(-) + CH(3)Y relative translational or CH(3)Y vibrational excitation are possible, e.g., the roundabout mechanism.     

基因网络前馈环路中的涨落共振

应用化学主方程和线性涨落近似方法,重点研究了前馈环路（FFL）对外界输入弱信号的响应,特别考察了它的涨落共振现象．研究发现Z基因的FR行为很大程度上依赖于FFL的协同性：协同FFL中Z的FR曲线呈明显的单峰,而非协同FFL中该曲线出现明显双峰．由于振荡信号常常在实际应用中用来探测网络的调控结构,因此可以利用涨落共振曲线的定性差别来区分FFL网络的性能．     

Substituent effects in cation-π interactions: a unified view from inductive, resonance, and through-space effects

The quantification of inductive (I), resonance (R), and through-space (TS) effects of a variety of substituents (X) in cation-π interactions of the type C?H?X···Na? is achieved by modeling C?H?-(Φ?)(n)-X···Na? (1), C?H?-(Φ?)(n)-X···Na? (2), C?H?-(Φ(2perpendicular))(n)-X···Na? (2'), and C?H? ···HX···Na? (3), where Φ? = -CH?CH?-, Φ? = -CHCH-, Φ(2perpendicular) indicates that Φ? is perpendicular to the plane of C?H?, and n = 1-5. The cation-π interaction energies of 1, 2, 2', and 3, relative to X = H and fitted to polynomial equations in n have been used to extract the substituent effect E?1, E?2, E?(2'), and E?3 for n = 0, the C?H?X···Na? systems. E?1 is made up of inductive (E(I)) and through-space (E(TS)) effects while the difference (E?2 - E?(2')) is purely resonance (E(R)) and E?3 is attributed to the TS contribution (E(TS)) of the X. The total interaction energy of C?H?X···Na? is nearly equal to the sum of E(I), E(R), and E(TS), which brings out the unified view of cation-π interaction in terms of I, R, and TS effects. The electron-withdrawing substituents contribute largely by TS effect, whereas the electron-donating substituents contribute mainly by resonance effect to the total cation-π interaction energy.     

Quinazoline Antifolate Thymidylate Synthase Inhibitors: Replacement of Glutamic Acid by Aminophosphonic Acids

The synthesis of six analogues of the potent thymidylate synthase (TS) inhibitor N -[4-[ N -[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinoyl)-methyl]- N -prop-2-ynylamino]benzoyl]- L -glutamic acid 2 is described in which the glutamic acid residue has been replaced by DL -aminophosphonic acids. New antifolates were tested as inhibitors of TS isolated from mouse L1210 leukemic cells as well as inhibitors of growth mouse leukemic L5178Y cells. In general these modifications result in compounds that are considerably less potent than 2 as TS inhibitors with K i 's 0.17-1.10 w M. Very poor solubility in water limited their proper assay of growth cells inhibition.     

Kinetics of aquation and anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts

The aqua adducts of the anticancer complexes [(eta(6)-X)Ru(en)Cl][PF(6)] (X=biphenyl (Bip) 1, X=5,8,9,10-tetrahydroanthracene (THA) 2, X=9,10-dihydroanthracene (DHA) 3; en=ethylenediamime) were separated by HPLC and characterised by mass spectrometry as the products of hydrolysis in water. The X-ray structures of the aqua complexes [(eta(6)-X)Ru(en)Y][PF(6)](n), X=Bip, Y=0.5 H(2)O/0.5 OH, n=1.5 (4), X=THA, Y=0.5 H(2)O/0.5 OH, n=1.5 (5 A), X=THA, Y=H(2)O, n=2 (5 B), and X=DHA, Y=H(2)O, n=2 (6), are reported. In complex 4 there is a large propeller twist of 45 degrees of the pendant phenyl ring with respect to the coordinated phenyl ring. Although the THA ligand in 5 A and 5 B is relatively flat, the DHA ring system in 6 is markedly bent (hinge bend ca. 35 degrees ) as in the chloro complex 3 (41 degrees ). The rates of aquation of 1-3 determined by UV/Vis spectroscopy at various ionic strengths and temperatures (1.23-2.59x10(-3) s(-1) at 298 K, I=0.1 M) are >20x faster than that of cisplatin. The reverse, anation reactions were very rapid on addition of 100 mM NaCl (a similar concentration to that in blood plasma). The aquation and anation reactions were about two times faster for the DHA and THA complexes compared to the biphenyl complex. The hydrolysis reactions appear to occur by an associative pathway. The pK(a) values of the aqua adducts were determined by (1)H NMR spectroscopy as 7.71 for 4, 8.01 for 5 and 7.89 for 6. At physiologically-relevant concentrations (0.5-5 microM) and temperature (310 K), the complexes will exist in blood plasma as >89 % chloro complex, whereas in the cell nucleus significant amounts (45-65 %) of the more reactive aqua adducts would be formed together with smaller amounts of the hydroxo complexes (9-25 %, pH 7.4, [Cl(-)]=4 mM).     

Alphabet‐Inspired Design of (Hetero)Aromatic Push–Pull Chromophores

Push–pull molecules represent a unique and fascinating class of organic π‐conjugated materials. Herein, we provide a summary of their recent extraordinary design inspired by letters of the alphabet, especially focusing on H‐, L‐, T‐, V‐, X‐, and Y‐shaped molecules. Representative structures from each class were presented and their fundamental properties and prospective applications were discussed. In particular, emphasis is given to molecules recently prepared in our laboratory with T‐, X‐, and Y‐shaped arrangements based on indan‐1,3‐dione, benzene, pyridine, pyrazine, imidazole, and triphenylamine. These push–pull molecules turned out to be very efficient charge‐transfer chromophores with tunable properties suitable for second‐order nonlinear optics, two‐photon absorption, reversible pH‐induced and photochromic switching, photocatalysis, and intercalation.

     

Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications

The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.     

Multiplex PCR with multichannel microchip electrophoresis: an ultrafast analysis for genetic diseases

A Y chromosomal polymorphic markers screening strategy using a multiplex polymerase chain reaction (PCR) and DNA microchip electrophoresis technology has recently been developed. It is a part of the human Y chromosome haplotyping system for studying Japanese population genetics and its relationship with male spermatogenic failure. This strategy is based on optimizing and modifying the primer set concentrations while keeping all other components of the PCR mixtures and conditions similar to those of a singleplex PCR. Well-balanced PCR products are obtained without changing even the DNA oligomer melting temperatures. Here, a panel of primer sets are used to amplify two groups of Y chromosome markers. The first consists of five markers and the second consists of seven markers. Both are possibly deleted in infertile men. The microchip electrophoresis technology is fast and sensitive, enables direct molecular typing of several Y chromosomal markers, and is separated by a difference of as many as six base pairs.