多菌落分组PCR法从高背景中筛选重组子

多菌落分组ＰＣＲ法从高背景中筛选重组子王翔俞炜源军事医学科学院生物工程研究所北京１０００７１在基因工程操作中，重组子的筛选、鉴定是必不可少的步骤，一般来说，这属于常规技术，不会有太大难度，但在很多情况下，人们会被很低的重组效率所困扰，如平端连接、部分...     

PCR在ABO疑难血型鉴定中的应用

ABO血型抗原是人类最主要的血型抗原，ABO疑难血型的鉴定正成为安全输血的一个重要课题。随着ABO血型基因的成功克隆，PCR分型技术的应用，使ABO型别分析达到了更精细的水平。本文对ABO疑难血型的鉴定现状、PCR鉴定的原理和方法进行了综述。随着分子生物学理论和实验技术的进一步发展，PCR方法在鉴定ABO疑难血型方面将有更广泛的应用前景。     

一种快速鉴定重组酵母克隆的方法

甲基营养型毕赤酵母巴斯德毕赤酵母(Ｐｉｃｈｉａｐａｓｔｏｒｉｓ)是目前较为有效的外源基因表达系统,可实现目的蛋白的高效分泌表达,且可对目的蛋白进行糖基化加工等[1]。毕赤酵母表达系统利用的是乙醇氧化酶基因(Ａｏｘ1)启动子作为强诱导型启动子,可被甲醇诱导。宿主菌ＧＳ115在Ｈｉｓ4基因位点被突变,因而只有当携有外源基因和Ｈｉｓ4基因的表达载体(如ｐＰＩＣ9或ｐＨＩＬＳ1)与酵母染色体在Ａｏｘ1基因处发生同源重组后,酵母克隆才能在组氨酸缺陷培养基(ＭＤ)平板上生长。而此时由于酵母基因组上Ａｏｘ1基因被取代,重组克隆仅能利用…     

用聚合酶链反应和限制性内切酶技术快速鉴定医学真菌

介绍了一种基于聚合酶聚链反应（ＰＣＲ）结合限制性内切酶分析技术区分临床重菌的方法，首先利用９种真菌的通用引物扩增其１８ＳｒＲＮＡ基因，然后用ＨａｅⅢＢｇｌＩ和ＮｈｅＩ三种限制性内切酶将扩增产物消化成不同数量和不同大小的ＤＮＡ片段，可以对白色念珠菌，新型隐球菌和烟曲霉菌等９种临床上常见的医学真菌作出快速鉴定，该技术具有快速，敏感，不需杂交，可在２４ｈ以内完成鉴定等优点，有良好的临床应用前景。     

普通PCR与TD-PCR在临床医学科研中应用价值的比较

目的 比较普通聚合酶链反应(PCR)与降落聚合酶链反应(Touchdown PCR,TD-PCR)在临床医学科研中的价值.方法 以人类外周血基因组DNA为模板,设计VHL基因3个外显子的3对引物,根据普通PCR及TD-PCR原理设计包括3个外显子片段在内的PCR程序,通过试验选择PCR最佳反应条件,在同一程序中分别对3个片段进行扩增,琼脂糖凝胶电泳检测扩增产物,纯化后PCR产物测序分析两种试验方法 的差别.结果 电泳检测及纯化后DNA产物测序分析均显示TD-PCR扩增产物条带特异性、效率较普通PCR扩增产物高.结论 成功建立了TD-PCR方法 ,TD-PCR方法 较普通PCR更为高效实用,为临床基因突变筛查研究提供了快速可靠的手段.     

毛细管PCR技术的建立及其在传染病快速诊断上的应用

毛细管ＰＣＲ技术的建立及其在传染病快速诊断上的应用李建标，谭津，赖进祥，王春颖，朱美财，林万明临床实验科主题词聚合酶链反应，传染病，基因聚合酶链反应（ＰＣＲ）技术的发展及其在传染病诊断上的应用，使传染病的基因诊断有了新的突破［１］。然而常规ＰＣＲ反应...     

用聚合酶链反应可准确鉴定移植前牛胚胎的性别

聚合酶链反应(PCR)技术是胚胎性别鉴定的一大突破。我们发展了先进的PCR技术,用于移植前牛胚胎的性别鉴定,用两个特异的雄性Y染色体重复序列为标靶,以PCR方法对DNA进行扩增。我们进一步扩增了出现在雄性和雌性中的牛重复序列。这一方法极其准确,结果无一差错,可用于90％以上的牛半胚性别鉴定。     

差别显示技术在寻找新基因中的应用

综述了应用ｍＲＡ差别显示技术寻找新基因的方法，应用及前景。     

PCR技术在病毒性疾病诊断中的应用

ＰＣＲ技术在病毒性疾病诊断中的应用武警医学院微生物教研室王丹敏综述于德芳审校（天津３００１６２）关键词聚合酶链式反应病毒性疾病聚合酶链式反应技术（ＰＣＲ）是８０年代中期出现的分子生物学技术,它可使靶ＤＮＡ序列在短时间内由极少量增加至上百万倍。由于具有...     

实时定量PCR阵列技术在规模化检测基因表达中的应用

实时定量PCR阵列技术（RQ-PCR-array）以实时定量PCR（RQ-PCR）技术为核心并结合阵列（array）技术,能够比较可靠、准确地检测信号转导、疾病相关通路等过程相关的基因表达图谱,在规模化检测基因表达方面具有高通量的显著优势,已广泛应用于基础研究和临床。如能够结合多重PCR技术,有可能进一步达到超高通量基因表达检测水平。本文对该技术在规模化检测基因表达中的研究进展进行综述。     

ABO genotyping by inverse PCR technique

Inverse PCR technique was applied to type three major alleles (A(1), B and O(1)) of the ABO blood group by simultaneously detecting separated allele-determining nucleotides (the 261st base in exon 6 and the 796th and 803rd nucleotides in exon 7) of the ABO gene. A sequence of about 1.7 kb from exons 6 to 7 of each allele was amplified, both termini of the fragment ligated, and allele-typing performed by the inverse PCR-restriction fragment length polymorphism (IP-RFLP) and allele-specific inverse-PCR (ASIP) methods. For intramolecular ligation, primers for the first PCR were designed to have Acc I-restriction sites within the sequences, and both termini of the 1.7-kb fragment were digested with Acc I. Using the IP-RFLP method, the inverse PCR product was digested with Kpn I, Nla III and Dde I, A(1), B, O(1)-standard (O(A)) and O(1)-variant (O(G)) alleles were detected as 365-bp, 272-bp, 193-bp and 128-bp fragments, respectively. By the ASIP method using four allele-specific primers, 222-bp, 124-bp and 232-bp fragments were amplified from A(1), B and O(1) templates, respectively. These techniques would be applicable to detecting separated polymorphic regions of some other genes.     

Magnetic resonance of the brain: the optimal screening technique

Magnetic resonance (MR) images and computed tomograms of 25 patients with head trauma were compared. MR proved to be superior in many ways for demonstrating extracerebral as well as intracerebral traumatic lesions. Isodense subdural hematomas, which present a diagnostic dilemma on CT images were clearly seen on MR, regardless of their varying CT densities. In a case of epidural hematoma, the dura mater was shown directly as nearly devoid of signal on MR. Direct coronal images provided excellent visualization of extracerebral collections along the peritentorial space and subtemporal area. In a patient with intracerebral hematoma, CT failed to demonstrate residual parenchymal changes in a 3-month follow-up study, but MR clearly depicted the abnormalities. The superiority of MR over CT was also well illustrated in a patient with post-traumatic osteomyelitis of the calvarium.     

Image features of true positive and false negative cancers in screening mammograms

The location, tissue background and imaging characteristics of true positive and false negative screens of breast cancers have been studied. This data can aid decisions in optimizing the display of mammographic information with the objective of minimizing false negative screens. Screening mammograms for four groups of women were digitized; those with screen detected cancers, those with false negative interval cancers, and matched normals for both groups. The optical density (OD) distribution in the main breast region of each mammogram was determined. The OD in three regions of interest around the cancers was also measured. Cancer locations were mapped and warped onto a typical image to show their spatial distribution. Where a cancer was detectable by calcifications alone it had a relatively low probability of being a false negative interval cancer. The mean OD differences between the cancer and the cancer background region (excluding calcifications) were approximately a factor of two lower in dense breasts compared with other breast types. Poorly defined masses that became interval cancers had mean OD differences that were approximately a factor of 0.1 OD lower than those that were detectable by screening. 22% of false negative cancers were located near the chest wall edge of the mammograms compared with 10% of the true positives. The results indicate the importance of effectively displaying information in the lighter areas of the mammogram, corresponding to glandular tissues, with sufficient contrast for suspicious mammographic details to be detected. Where the mean OD differences between the cancer and its background region are low, as measured for some poorly defined masses, there is an increased risk of a false negative interval cancer. Particular attention should be given to the chest wall area of the film, especially in the lower retroglandular region, during routine screening.     

Screen-film mammographic technique for breast cancer screening

The values of dose and signal-to-noise ratio (SNR) for many techniques and breast thicknesses were computed and compared with a reference technique and breast thickness to provide a valid basis upon which to select a screening technique for screen-film mammography. The reference consisted of a Min-R/OM1 combination (or its equivalent) exposed through a 4.5-cm thick breast via 28 kV and a molybdenum target-beryllium window tube with a 0.03-mm Mo filter. Radiographs of an improved breast phantom were used to relate computed relative SNR values of techniques to diameters of the smallest calcific and soft-tissue objects demonstrated in mammograms. Without use of a grid, four Mo target/filtration combinations yielded similar computed dose and SNR levels, as did a tungsten target tube operated at 23 kV without a filter. Operation of the latter tube at 27.5 kV with a 0.051-mm rhodium filter should reduce dose by half but also SNR by 22%. However, such operation with a grid should greatly improve imaging moderate to large breasts without increasing dose over reference values.     

Modular real-time PCR screening assay for common European animal families

A screening assay based on real-time PCR and melt curve analysis was developed to detect DNA from nine common European animal families/species and human. The assay consists of a 10-cycle universal pre-amplification followed by specific nested PCR and was designed to exploit the different melting temperatures (T _m) of family/species-specific 12S ribosomal ribonucleic acid and cytochrome b fragments, which are amplified in duplex reactions. Case-related modular application is possible. Beyond determination of the animal family and discrimination from human DNA, evaluation of the melt curve in some cases additionally allows for species determination (e.g. cat vs. lynx). The method presents a quick, flexible and sample-saving approach to assess non-human DNA at low expenses, and it is especially useful in resolution of DNA mixtures.     

Avoidable surgical consultations in women with a positive screening mammogram: Experience from a southern region of the Dutch breast screening programme

Introduction

According to current Dutch guidelines, all women with a positive screening mammogram are referred for a full hospital assessment, which includes surgical consultation and radiological assessment. Surgical consultation may be unnecessary for many patients. Our objective was to determine how often surgical consultations can be avoided by radiological pre-assessment.

Materials and methods

All women with a positive screening mammogram, referred to our radiology department between 2002 and 2007, were included (n = 1014). Percentage of women that was downstaged to BI-RADS category 1 or 2 by radiological pre-assessment was calculated. Negative predictive value (NPV) for malignancy was estimated from the in-hospital follow-up, which was available up to September 2012.

Results

423 of 1014 women (42%) were downstaged to BI-RADS category 1 or 2 by radiological pre-assessment. During follow-up, 8 of these 423 women (2%) developed a malignancy in the same breast. At least 6 of these malignancies were located at a different location as the original screening findings which led to the initial referral. The estimated NPV for malignancy was 99.5% (95%CI, 98.3–99.9).

Conclusion

By referring women with a positive screening mammogram to the radiology department for pre-assessment, a surgical consultation was avoided in 42%, with an estimated NPV of 99.5% for malignancy.     

Computer-simulation technique for low dose computed tomographic screening

OBJECTIVES: The purpose of this study is to assess the relative influence of noise and artifact in detecting lung nodules on low dose computed tomographic (CT) screening. METHODS: We develop the computer-simulation technique that allows tube current simulation and virtual nodule insertion in any CT images. The tube current simulation uses a reduction model that adds random Gaussian noise distribution to existing projection data. The virtual nodules are generated using a dedicated CT simulation tool with same scanner geometry. RESULTS: The coefficient of the correlations between the contrast-to-noise ratio of the actual scan and simulated tube current images was 0.98. There was no difference in CT number between virtual nodules and actual nodules [t test results = 0.60, t50(0.01) = 2.70 at 10 mA] and the coefficient of the correlations of the image noise was 0.99. CONCLUSIONS: Our technique is useful for systematic evaluation of radiation dose reduction and structure visibility in low-dose CT screening.     

PCR快速筛选质粒cDNA文库方法的建立

目的　建立一种快速、有效地筛选质粒ｃＤＮＡ 文库的方法—ＰＣＲ 法。方法　将质粒ｃＤＮＡ文库重组子进行矩阵排列,然后用特异引物进行ＰＣＲ 逐级筛选以获得目的克隆。结果　经两轮筛选（约１ 周的时间）获得４ 株阳性克隆。结论　ＰＣＲ 矩阵筛库法不仅适用于常规ｃＤＮＡ 克隆的筛选,还适用于筛选较短的ｃＤＮＡ片段,比常规的筛库方法更为灵敏、特异、高效。