A Simple and Efficient Method for DNA Purification from Samples of Highly Clotted Blood

Rapid purification of DNA from samples of highly clotted blood is a challenging problem due to the difficulty in recovering and dispersing blood clots. We developed a new method for discarding the serum-separator gel and rapidly dispersing the blood clots. A special disposable tip was inserted into the serum-separator gel so that the serum-separator gel could be discarded. The blood clot obtained was dispersed into small pieces through a copper mesh (pore size, 250 μm) in a special dispersing instrument by centrifugation. After lysis of red blood cells and white blood cells, genomic DNA was concentrated and desalted by isopropanol precipitation. The mean yield of DNA purified from a 0.3-ml blood clot was 22.70 μg in 173 samples of clotted blood cryopreserved for 1 month, and 19.02 μg in 1,372 samples of clotted blood cryopreserved for >6 months. DNA samples were successfully performed through polymerase chain reaction, real time polymerase chain reaction, and melt curve analysis. Their quality was comparable with that purified directly from EDTA-anticoagulated blood. The new method overcomes the difficulties in recovering and dispersing blood clots, allowing efficient purification of DNA from samples of highly clotted blood.     

一种从人血凝块中提取基因组DNA的方法

介绍了一种新的从人血凝块中提取基因组DNA的方法,用该方法提取的DNA成功地应用于PCR和限制性酶切等后续实验中。基本过程为首先机械粉碎,然后高盐高EDTA溶液处理,含蛋白酶K和SDS的消化液变换温度消化,苯酚氯仿抽提。     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions,

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Highlights

• •Endogenous plasma/serum QC marker that quantifies cumulative sample thawed time.
• •Mechanism of marker change known, and rate law established.
• •Data interpretation based on documented population averages and rate law.
• •Blind-challenge verified; utility proven via exposure of undisclosed freezer outage.

     

一种改进的分离嗜中性白细胞的方法

报道一种从全血中分离嗜中性白细胞的方法.人血经过葡聚糖沉降,淋巴细胞分离液梯度分离,特殊分离液的洗涤和红细胞的溶胀后,得到了嗜中性白细胞.经瑞特氏染色和台盼蓝染色证明细胞纯度与存活率均在95％以上.以化学发光和细胞色素c还原法证明了细胞的高活力与膜受体的完整性.与国外的分离方法相比较,该方法简便易行,且分离效果好,是一种高效、经济的分离方法.     

A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H₂O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.     

A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein

The in vitro extraction of cholesterol from erythrocytes by plasma lipoproteins of reduced cholesterol content would be expected to be free of cholesterol-unrelated alterations of the cell membrane. The earlier application of this method utilized whole blood plasma in which the major part of the lipoprotein cholesterol was esterified by the plasma enzyme lecithin-cholesterol acyl transferase (LCAT) in a preliminary incubation. Because of the cholesterol remaining unesterified in the plasma, only 35% of the cell cholesterol could be removed. The method reported here uses HDL., a plasma lipoprotein which is the preferred substrate for LCAT, instead of whole plasma for the extraction. Multiple extractions with LCAT treated HDL, resulted in the removal of up to 77% of the erythrocyte cholesterol with only minor hemolysis.     

A Simple Method for Selective Isolation of Stenotrophomonas maltophilia from Environmental Samples

Stenotrophomonas maltophilia is a commonly found environmental bacterium that is associated with the plant rhizosphere. It shows increasing prevalence in immunocompromised patients. We report a simple method for selective isolation of S. maltophilia from soils which makes use of both its resistance to imipenem and its requirement for methionine.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

A Method for Separation of Granulocytes From Normal Human Blood Using Hidroxiethrl Starch

A comparison of the granulocyte yield obtained from normal buffy coats following the addition of different concentrations of hydrojtyethyl starch (H.E.S.) was made. Hydrcocyethyl starch proved to be at least as effective as Dextran both in regard to the percentage of granulocytes recovered and the speed of sedimentation of the red blood cells.     

人TRAP1基因的克隆、原核表达及表达条件优化

应用RT-PCR技术,从人白血病多药耐药细胞株K562/ADR中扩增出肿瘤坏死因子受体相关蛋白l(tumor necrosis factor receptor-associated protein1,TRAP1)基因的cDNA.选择Nde I和Xho I分别作为上下游引物的酶切位点,将TRAP1基因克隆到带有6×His标签的pET-28a(+)的载体上.重组质粒转化大肠杆菌DH5α中,涂布于含卡那霉素的LB琼脂培养基上,经双酶切鉴定后的阳性克隆送去测序.测序成功的重组质粒pET28a(+)-TRAP1转化大肠杆茵BL21 (DE3)中,在IPTG的诱导下,成功表达重组蛋白TRAP1.通过改变诱导温度、诱导时机、IPTG浓度及诱导时间,找出最佳表达条件,使重组蛋白TRAP1表达量最高.结果表明,在39℃条件下,0D600达到0.8时,经终浓度为0.1 mmol/L的IPTG诱导6h,目的蛋白的表达量最高.该研究为纯化出TRAP1蛋白,进一步研究该蛋白的结构和功能奠定了基础.     

一种快速、经济提取外周凝血基因组DNA 方法的建立

目的:建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法:摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用KI法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量,并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的KI法进行比较分析。结果:最佳的匀浆条件为:39000 rmp,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论:与常规的外周凝血提取方法相比(蛋白酶K消化法),本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

一种快速、经济提取外周凝血基因组DNA方法的建立

目的：建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法：摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用Ⅺ法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量。并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的Ⅺ法进行比较分析。结果：最佳的匀浆条件为：39000map,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论：与常规的外周凝血提取方法相比（蛋白酶K消化法）,本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

X-Ray Small Angle Scattering: A New Deconvolution Method for Evaluating Electron Density Distributions from Small Angle Scattering Diagrams

The direct determination of the electron density distributions of multilayered specimens with a small number of unit cells from X-ray small angle scattering experiments via the Q-function method of Hosemann and Bagchi includes the deconvolution of the so-called Q_o-function, the generalized Patterson function of one unit cell. In this paper a new and direct deconvolution method on the basis of Fourier series is presented which is suitable for one-dimensional centrosymmetrical (or antisymmetrical) density distributions. A FORTRAN-program has been written which has an execution time of ca. 20 s on an UNIVAC 1106-computer. The procedure has been successfully tested on some convolution functions generated by membrane-type electron density distributions.     

从血液中提取总RNA的一种快速高效方法

血液中含有大量的RNA酶 ,可引起RNA的降解 .防止RNA酶的降解 ,是保证所得RNA片段完整的关键 .目前提取RNA的方法较多 ,但有些方法尚不能完全防止RNA降解 .将TRIZOL方法稍加改进 ,将TRIZOL与异硫氰酸胍联用提取血液淋巴细胞总RNA .琼脂糖凝胶电泳结果表明 ,其 2 8SRNA与 18SRNA的比值为 2∶1,优于单独使用其中任何一种试剂者 .此方法同样适用于从其它细胞中提取RNA .