Urine as another potential source for template DNA in polymerase chain reaction (PCR)

This technical note examines the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred μl of urine yielded a template DNA for PCR. This research will increase the opportunity for scientists to examine the genetic backgrounds of their target animals by using non‐invasive sample collection in the wild. Am. J. Primatol. 48:299–304, 1999. © 1999 Wiley‐Liss, Inc.     

Use of DNA from dry leaves for PCR and RAPD analysis

Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA. Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis.     

PCR检测血清HBV DNA

本文利用ＨＢＶ ＤＮＡ基因组ｐｒｅＣ和Ｃ区的一套引物,以ＰＣＲ法检测了２６例健康正常人血清和９５例免疫标志各异、临床症状不同的乙肝或可疑乙肝患者的血清,前者无１例检出ＨＢＶ ＤＮＡ,后者共检出的６６例血 清存在ＨＢＶ ＤＮＡ。该方法特异性强,适用于检测任一亚型的ＨＢＶ ＤＮＡ,一轮ＰＣＲ最低可检出０．１ｐｇ的ＨＢＶ ＤＮＡ。     

应用PCR技术定向引入DNA小片段的策略

描述一种应用PCR技术定向引入DNA小片段和特异酶切位点的方法。为了获得m2/loxp66EGFPloxp71基因片段。根据EGFP基因序列,设计一对特异引物,上、下游引物分别引入m2/loxp66、loxp71序列和Xhol、Mlu1酶切位点。以pEGFPN1质粒为模板,采用PCR扩增以合成DNA双链,插入到克隆载体pMD18T。对重组子测序结果表明,实现了DNA小片段和酶切位点的定向引入。     

水稻PCR扩增模板的快速制备

用碱处理水稻嫩叶,取碱处理水稻叶片浸出液直接用作PCR扩增的模板,扩增结果稳定、准确,与常规提取的DNA扩增效果相比没有显著差异。用此方法制备的模板室温下2周之内、4℃下3周之内、-20℃下4个月以上扩增结果不变。此方法所需试剂均为常规试剂,具有需要材料少、成本低、简便、快捷等优点,尤其适合材料比较宝贵的转基因水稻的预筛选和大样本量的PCR检测,为分子标记技术的实际应用创造了条件。 Abstract:The solution of alkali-treated fresh rice leaves was used directly as the templates of PCR.The amplified results were stable,reliable,and had no difference compared with that amplified with rice total DNA extracted by common method.The stable results can still be obtained based on the templates kept at 25℃ for tow weeks,at 4℃ for three weeks,at -20℃ for over four months.With this technique,less material and only common reagent are required,which is especially adapted to the screening of the precious trangenic rice in advance and large-scale PCR tests.     

Comparison of small-scale methods for the rapid extraction of plant DNA suitable for PCR analysis

We present results from a comparison of six methods for rapid DNA extraction from leaf and other plant tissues. We have used samples from six plant species in our study, including both crop species and their wild relatives. The success of the methods is assessed by PCR of the DNA using conserved primers, and the applicability of the different methods to particular species and tissues is assessed. The speed, reliability, convenience, and potential for further improvement of the methods are also discussed.     

定量PCR的非同源对照模板的构建

建立了HCV RNA和hTERT mRNA的竞争定量PCR系统。对照模板的构建方法是：利用计算机辅助优选设计连接引物,低严紧型扩增大肠杆菌DNA,回收并克隆预期的DNA片段。该片段与靶序列除两端引物序列完全相同外,无同源性,因此可作为非同源对照模板,这种构建同源对照模板的方法,简便易行,适应面广。     

古代生物遗迹中DNA的PCR分析

古代生物遗迹中ＤＮＡ所反映出的遗传信息正成为古生物学家研究生物进化的越来越重要的资料,而ＰＣＲ则是揭示古ＤＮＡ遗传信息的最简便和重要的方法。本文评述了古ＤＮＡ进行ＰＣＲ分析的有关方法、存在的问题及实验结果的一些甄别标准。     

Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA

Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland–Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.     

通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

利用PCR法检测HIV—1DNA

通过比较多个HIV(人免疫缺陷病毒)分离株的核苷酸序列,我们选择膜基因上7373—7514位的一段保守区为目的片段合成了引物1(5′—AGCAGCAGGAAGCACTATGGGC—3′)和引物2(5′—CCAGACTGTGAGTTGCAACA—3′),并分别以质粒PⅢexE7和MT4-HIV-1 DNA,为模板进行了PCR反应及敏感性试验。结果表明,用PCR法可检测出1～10个质粒分子及1×10~6个细胞中一个感染细胞。因此我们推论,本法可应用于AIDS临床标本的检测。     

用于PCR扩增的DNA简易制备法现状

介绍了 6种用于PCR扩增的DNA简易制备法。所得DNA质量符合PCR扩增之用 ,经过试验也可用于系统生物学之研究     

一种高特异性的改良降落PCR

为提高基因组DNA中的基因PCR检出的特异性,设计了一种改良的降落PCR程序,并分别用TaqDNA聚合酶及高保真PfuDNA聚合酶进行实验。自盐藻Dunaliella bardawil中提取基因组DNA作为PCR模板,使用TaqDNA聚合酶及PfuDNA聚合酶,运用普通PCR和降落PCR程序,扩增胡萝眩素生物合成相关基因（cbr）上游启动子序列,并电泳比较PCR扩增产物的特异性。结果显示,使用普通Taq酶PCR,普通PCR程序产生200bp,500bp和1272bp长的三条带,而TD-PCR程序仅克隆出1272bp的特异带;利用高保真的PfuDNA聚合酶作PCR,在TD-PCR泳道中仅有1272bp一条带,而普通PCR除了1272bp的特异带外,还出现一条500bp的非特异带。无论使用普通Taq酶或高保真酶Pfu,改良的降落PCR程序均明显提高PCR的特异性,类似的降落PCR程序可望用于克隆用普通PCR难以克隆的基因片段,或在假阳性难以去除的情况下提高PCR的特异性。     

高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Fast preparation of fungal DNA for PCR screening

Rapid DNA preparation for the quick screening is highly demanded in diverse research fields. Here, we combined an extraction buffer and heat treatment to generate DNA templates from yeast and filamentous fungal materials for PCR. This method may be widely applicable to diverse fungal species in clinical and basic studies.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

国产HBVDNAPCR试剂的质量研究

首次利用中国药品生物制品检定所建立的一套国家参考品对国内几个厂家的ＰＣＲ试剂进行检测,检测过程中发现这几家国产ＰＣＲ试剂都是利用原血清直接扩增ＨＢＶＤＮＡ,比较快速,且灵敏度较高,但也存在一些问题需要更深入的研究。同时对这几家ＰＣＲ试剂的临床应用价值做了讨论,并讨论了应注意的几个问题     

PCR检测伪狂犬病病毒DNA

 根据伪狂犬病病毒 (PRV)gB基因的序列 ,设计并合成了一对引物 ,以闽A株细胞培养毒为模板 ,筛选最佳反应条件 ,建立了检测PRV的PCR方法 应用该方法对Fb、Bartha、BJ、GD、V2F4、S、S3、SR、Buk、Shope、Norden、MinkⅢ、HB、F8、F9、F12等毒株的细胞培养液进行基因扩增 ,均获得了分子量为 2 81bp的特异性目的DNA片段 ,而对Vero细胞与FMDV、SVDV、HCV、PRRSV、JEV、PPV等病毒进行检测 ,结果均为阴性 ,没有出现交叉反应 对PRV毒株扩增的产物测序 ,结果序列与文献报道一致 ,证明PCR扩增产物和方法的特异性 对 1994～ 2 0 0 0年期间送检的临床样品和保存的PRV毒种 ,用病毒分离、双抗体夹心ELISA和PCR等 3种方法进行检测 ,结果前 2种方法检测为阳性的 ,PCR检测均为阳性 ;PCR检测为阴性 ,前 2种方法检测也为阴性 ;可是 ,前 2种方法检测为阴性的 ,PCR却检测出部分阳性 ;经x2 检验 ,证明PCR检出率明显高于前 2种方法的检出率 对PRV闽A株细胞毒提取物DNA进行检测 ,其最低检出量为 15 8pg 对 1999～ 2 0 0 0年期间广东、福建、海南等省的 31个大中型猪场送检的 191份病料进行检测 .结果病料阳性率为 2 6 2 % ( 50 191) ,猪场阳性率为 71% ( 2 2 31) 实验结果表明 ,所建立的PCR技术可用于伪狂犬病的快速诊断