A general method for isolation of high molecular weight DNA from eukaryotes

A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.     

The flexibility of low molecular weight double-stranded DNA as a function of length. I. Light scattering measurements and the estimation of persistence lengths from light scattering, sedimentation and viscosity

In the preceding paper are described the isolation and physical characterization of seven narrowly disperse fractions of calf thymus DNA in the molecular weight range 0.3 to 1.3 X 10(6) daltons. Herein, we have determined by light scattering the molecular weights and root mean square radii of these fractions in a solvent comprising 0.2 M NaCl, 2 mM EDTA, 2mM Na-PO4,pH7. Measurements were made in a modified Wippler-Scheibling photometer to a 20 degree lower limit of scattering angle on solutions rendered virtually dust-free by procedures described. The optical anisotropies of the DNA fractions were measured permitting the experimental molecular weights and root mean square radii to be corrected to their true values. From these values, with appropriate polydispersity corrections, we calculate a Kratky-Porod persistence length, a, of 54.0 +/- 5.6 nm which is invariant over the molecular range examined. From the sedimentation coefficients (preceding paper) and the theory of Yamakawa and Fujii, we calculate a to be 66 nm, a value found to apply equally well to several DNA samples of various origins whose sedimentation rates are known in themolecular weight range from about 4 X 10(4) to 10(8) daltons. Similarly, from the intrinsic viscosities and the theory of Yamakawa and Fujii, we calculate a to be 59 nm, which again adequately applies to a number of DNA samples whose viscosities have been measured by other workers in the molecular wieght range 3 X 10(5) to 10(8) daltons. The Flory-Mandelkern paramerter, beta, was found to vary with molecular weight in the manner predicted by the theory of Yamakawa and Fujii. The average value of a from the three sets of measurements is 60 +/- 6nm, which we believe applies to double-stranded DNA molecules, independent of chain length, over the whole range of molecular weights from which reliable data exist.     

A small angle light scattering device for planar connective tissue microstructural analysis

The planar fibrous connective tissues of the body are composed of a dense extracellular network of collagen and elastin fibers embedded in a ground matrix, and thus can be thought of as biocomposites. Thus, the quantification of fiber architecture is an important step in developing an understanding of the mechanics of planar tissues in health and disease. We have used small angle light scattering (SALS) to map the gross fiber orientation of several soft membrane connective tissues. However, the device and analysis methods used in these studies required extensive manual intervention and were unsuitable for large-scale fiber architectural mapping studies. We have developed an improved SALS device that allows for rapid data acquisition, automated high spatial resolution specimen positioning, and new analysis methods suitable for large-scale mapping studies. Extensive validation experiments revealed that the SALS device can accurately measure fiber orientation for up to a tissue thickness of at least 500 microns to an angular resolution of approximately 1 degree and a spatial resolution of +/-254 microns. To demonstrate the new device's capabilities, structural measurements from porcine aortic valve leaflets are presented. Results indicate that the new SALS device provides an accurate method for rapid quantification of the gross fiber structure of planar connective tissues.     

罗丹明6G共振光散射法测定废水中铊

在硫酸介质中, 痕量铊(Ⅲ)与碘化钾反应生成I₃^-, I₃^-与罗丹明6G形成1∶1缔合物, 可导致共振光散射明显增强, 据此建立了共振光散射测定痕量铊的新方法。考察了它们的光谱特征:罗丹明6G溶液的共振荧光峰波长为540 nm, (Rh6G-I₃)n缔合微粒共振散射峰波长为330、420、580 nm。通过条件试验确定0.4 mL 0.2 mol/L硫酸作为反应介质、0.1 mol/L KI溶液用量为0.4 mL、1.0×10^-4 mol/L罗丹明6G 溶液用量为0.4 mL、反应时间为5 min。进一步考察发现, 在580 nm波长, 共振散射光强度增加值与溶液中铊浓度呈线性关系, 方法的线性范围为0.005～0.10 mg/L, 检出限为0.001 2 mg/L。方法应用于工业废水中铊含量的测定, 结果与ICP-MS法的对照结果基本一致, 相对标准偏差(RSD, n=6)为1.8%～3.2%。     

共振光散射技术在无机分析中的应用

共振光散射技术是近年发展起来的一项简便灵敏的分析技术,其分析测定在一台普通的荧光光度计上就可加以实现。本文简要介绍了该项分析技术及其实验方法;归纳了该技术在无机分析中所常用的碱性染料体系、酸性染料体系、碘化物-碱性染料体系、表面活性剂体系等各种缔合物体系以及在难溶化合物体系、单质、溶胶或纳米颗粒体系等分析测定体系的应用;对利用二级散射技术、反二级散射技术,特别是共振瑞利散射技术测定30多种无机离子的方法体系及其应用情况进行了综述;在方法的应用范围、测定体系及理论研究等方面提出了合理的建议,旨在使该技术进一     

A simple method for site-directed mutagenesis with double-stranded plasmid DNA

Chromosome painting with library DNA probes specific for all human chromosomes was used to study the chromosomal content of micronuclei (MN) in normal and 5-azacytidine (5-aza-C)-treated lymphocyte cultures. More than 60,000 normal lymphocytes were screened for associated MN after in situ hybridization. At least 50 MN were scored for each probe. With the exception of chromosomes 12 and 19, which did not occur in MN, all other chromosomes were detected in MN at frequencies varying from 1 to 11.5%. Treatment of lymphocyte cultures with 5-aza-C induced preferential exclusion of chromosomes 1 (34%), 9 (32%) and 16 (20%) material in MN, whereas chromosome 8, 10, 12-15 and 21 material was not detected in MN. The results obtained from normal lymphocytes allow for the first time an estimation of the frequency of occurrence of all chromosomes in spontaneously occurring MN in human cells. Data derived from 5-aza-C-treated lymphocytes are furthermore consistent with the view that undermethylation of heterochromatin may be associated with loss of specific chromosomes at metaphase.     

A simple method for extracting DNA from old skeletal material

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.     

由两层散射体引起的声学散射问题的分裂方法

考虑由一个可穿透的两层散射体引起的声学散射问题.首先,利用位势理论和Fredholm理论,用积分方程证明了正散射问题解的存在性和唯一性.其次,根据散射问题解的积分表示,在散射体的外层介质内,波场由2部分组成,分别对应于散射体的2个界面引起的反射波.基于此,将两层散射体的散射问题分裂成2个耦合的经典透射问题,且证明了该分裂是唯一存在的.为了根据此分裂求得原问题的解,提出了一种解耦的迭代格式,每一步迭代只需要求解2个标准的透射问题,降低了计算的复杂性以及对计算机的存储要求.