A fluorescence-based sequence-specific primer PCR for the screening of HLA-B(*)57:01

Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA-B(*)57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA-B(*)57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence-specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non-infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence-specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method.     

DNA profiling of shahtoosh

The population of the Tibetan Antelope (Pantholops hodgsonii) has recently declined dramatically due to the illegal trade in its wool. The animal lives at high altitude and is protected from the extremely cold climate due to a coat of very fine wool. These hairs are highly sought for weaving a shawl called shahtoosh. The large-scale poaching of the antelope has resulted in the species being placed on CITES Appendix I. We report on a method of DNA profiling on a confiscated shahtoosh using the cytochrome b (cyt b) gene to produce a test that will identify unambiguously the presence of P. hodgsonii. Two shahtoosh samples were provided by the Council of Agriculture, Taiwan, and ten shawl samples of sheep wool (Ovis aries), cashmere from the Kashmir goat (Capra hircus), and pashmina from the Himalayan goat (C. hircus) were collected from local stores for comparison. Two primer pairs were used to amplify a 271 bp fragment of cyt b gene which would distinguish these three species. The resulting amplification products were directly sequenced. When the DNA sequences were compared with the sequences registered in GenBank and EMBL databases, the sequences with the highest homology were the cyt b genes of P. hodgsonii, C. hircus, and O. aries. This study demonstrates that there is still sufficient DNA present in the finished wool of a shahtoosh shawl to allow DNA typing and the method established was highly plausible to identify the CITES protected species.     

Intramolecular controls for the generation of clinical-quality SNP genotypes and the assessment of normal heterozygote imbalance

We present an inventive method that generates intramolecular controls for SNP analysis, termed Mirror SNPs. Using the ovine diseases of callipyge and scrapie as examples, we describe the PCR-driven production of balanced heterozygote copies of the various SNPs implicated in these diseases. In the absence of a callipyge-positive control DNA, we generated a balanced heterozygote Mirror SNP that represents both the wild-type and mutant forms of the causal polymorphism. Simultaneous analysis of this artificial Mirror SNP and the Real (target) SNP was used to prove the absence of the mutant form of the nucleotide at the Real SNP position in tested samples. Scrapie susceptibility was assessed using a PCR-driven system which generated four separate Mirror SNPs, and these enabled the confirmation of an apparent departure from 'balanced' heterozygote appearance at Real SNPs tested. Mirror SNP technology is generic and will enable the accurate assessment of rare and medically important SNP variants, more accurate frequency determinations, and the potential assessment of SNPs in 'mixed template' samples common in forensic analyses.     

A multiplex assay with 52 single nucleotide polymorphisms for human identification

A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously only partial STR profiles had been obtained. A total of 700 individuals from Denmark, Greenland, Somalia, Turkey, China, Germany, Taiwan, Thailand and Japan were typed, and the allele frequencies estimated. All 52 SNPs were polymorphic in the three major population groups. The mean match probability was at least 5.0 x 10(-19) in the populations studied. Typical paternity indices ranged from 336 000 in Asians to 549 000 in Europeans. Details of the 52 SNP loci and population data generated in this work are freely available at http://www.snpforid.org.     

跑道型结构光子晶体波导定向耦合器

鉴于波导定向耦合器在集成光路以及光电集成方面的广泛应用,提出了一种基于光子晶体波导间高效耦合的光子晶体定向耦合器。通过主波导和耦合波导间的耦合,可以实现对波长为1 490 nm和1 550 nm电磁波的高效分光。在将器件长度控制在30 μm左右的同时,其总效率高达93.05%。另外,发现主波导和耦合波导间介质柱结构参数对电磁波的耦合周期有着极大的影响。并通过将介质柱沿z方向拉伸0.1a(a为晶格周期),设计了工作波长为1 530 nm和1 540 nm的光子晶体定向耦合器,器件长度仅为60 μm。通过拉伸介质柱的纵向长度,可以大幅减小耦合周期,这对缩小器件体积以及实现更为密集的波分复用有着重要的意义。     

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit

DNA extracted from forensic samples can be degraded and also contain co‐extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpF?STR^® SGM Plus^® kit. The co‐amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX‐labelled primers. Co‐amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.     

SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION

We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.     

反射式多路投影立体显示系统的研究

设计了一种反射式多路投影立体显示系统,并介绍了该系统的成像原理及特性。系统将12幅视差图同时投影在柱面栅上,观察者仅凭肉眼就可看到具有双眼视差和运动视差的立体图像。系统成像视域较大,可以同时供多人观看。     

Multiplexed concentration quantification using isotopic surface‐enhanced resonance Raman scattering

The recently developed isotopically edited internal standard approach for surface‐enhanced resonance Raman scattering (SERRS) based chemical quantification is extended to demonstrate multiplexed detection of four different isotopic variants of a single chromophore. More specifically, it is shown that rhodamine‐6G (R6G) with 0, 2, 4, or 6 deuterium substitutions may be reliably quantified in either two‐ or three‐component mixtures. Thus, one isotopic species of known concentration may be used as an internal standard to determine the concentrations of two other isotopic components in a mixture. The concentrations of isotopic R6G SERRS chromophores are determined using partial least squares calibration and shown to yield a predictive accuracy of about ± 10% of the total R6G concentration (over 1–50 nM concentration range). These results set the stage for the use of such isotopic variants as tags for the SERRS/SERS quantitation of mixtures containing proteins, peptides, and other compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

人白细胞介素10(hIL10)在毕赤酵母中的表达

利用PCR技术从质粒pcDNA3．1／GS扩增得到hIL10基因．将其插入含有AOX1启动子和α分泌信号肽序列的毕赤酵母表达载体pPIC9K中，构建重组质粒pPIC9K／IL10，转化毕赤酵母GS115，筛选出整合了多拷贝白细胞介素10基因的菌株，经摇瓶培养，0．5％甲醇诱导表达、SDS-PAGE分析显示，表达产物以可溶性分子形式存在于上清中，诱导4d的表达量达到高峰．Western印迹表明．表达产物具有良好的抗原性和特异性．