生物素掺入PCR产物的反向杂交技术分析HLA基因型

本文介绍了与常规生物素或荧光标记引物不同,以及与同位素掺入ＰＣＲ产物不同的反向杂交技术,研究了生物素最佳掺入条件,测出加尾探针联结到尼龙膜上所需紫外光的强度,比较了生物素５＇端标记引物与生物素掺入ＰＣＲ产物的显色灵敏度,并根据实验测得的最佳条件分析了３个标准细胞株ＨＬＡ－ＤＰＢ１基因型。     

原位杂交及原位PCR检测幽门螺杆菌

本研究建立了检测幽门螺杆菌的原位杂交和原位ＰＣＲ技术,采用ＰＣＲ掺入的方法标记原位杂交的生物素探针,６份ＨＰ阳性胃活检组织冰冻切片杂交均为阳性,６份阴性对照组织为阴性。９／１２份ＨＰ阳性对照组织石蜡切片杂交阳性,２份阴性对照切片为阴性。３份阳性对照猫胃粘膜冰冻切片杂交也是阳性。原位ＰＣＲ的引物来自ＨＰ的１６ｓｒＲＮＡ基因,在扩增过程掺入生物素基因。４／４例ＨＰ阳性人胃粘膜冰冻切片原位扩增阳性,２／     

Nested PCR在检测牛白血病病毒前病毒中的应用

用ＮｅｓｔｅｄＰＣＲ检测牛白血病病毒的前病毒形式。从牛白血病病毒基因ｇｐ５１上选择两对引物进行ＮｅｓｔｅｄＰＣＲ体外扩增,产物经２％ａｇａｒｏｓｅ凝胶电泳和用生物素标记的探针鉴定,证实其特异性。结果表明该方法能从两个ＢＡＴ－ＢＬＶ细胞内检测出ＢＬＶ的前病毒ＤＮＡ形式。     

PCR和生物素探针对HFRSV的检测和分型的探讨

分析比较肾综合征出血热病毒（ＨＦＲＳＶ）７６／１１８株和Ｒ＿（２２）株的核苷酸序列,根据引物设计原则及检测分型的目的,设计并合成了３对引物。１对引物取于两毒株间的高同源区段,作为共同引物和外引物;另两对引物取于两毒株间的低同源区段,分别作为野鼠型引物、家鼠型引物和内引物。建立了ＤＮＡ聚合酶链反应（ＰＣＲ）和ＮｅｓｔＰＣＲ方法,并用ＮｃｓｔＰＣＲ合成了两种型特异的生物素探针。ＰＣＲ检测７６／１１８、Ａ＿９、陈、Ｒ＿２、Ｒ＿２２５个毒株,用外引物时均扩增出１条约３００ｂｐ的条带;用内引物的野鼠型引物时,除Ｒ＿（２２）株之外,其余４株均扩增出１条约７０ｂｐ的条带。斑点杂交试验证实了ＰＣＲ检测分型的准确性。ＮｅｓｔＰＣＲ和生物素探针斑点杂交试验可以测出１－１０ｂｇ的目的ｃＤＮＡ。     

应用套式PCR检测猪细小病毒

从猪细小病毒基因组的ＶＰ２基因上,选择两对引物进行套式聚合酶链反应体外扩增,产物经２％ａｇａｒｏｓｅ凝胶电泳和生物素标记的探针鉴定。证实具有特异性,敏感性实验证明,大套式ＰＣＲ能检测到１ ＴＣｉＤ５０的病毒量,在临床上应用方法能从不同的组织样品和粪便中检测出猪细小病毒,具有很高的敏感性。     

基因标记、转化、表达与检测

920468采用生物素和一种荧光染料标记的引物对聚合酶链反应产物进行定t分析〔英万Landgraf,A.“·/A rtal.Bioehem。一1991,193(2)一231~235〔译自DBA,1991,10(12),91一06628〕 经生物素和异硫氰酸荧光素共价标记的PCR引物可被固定化分离。采用在5‘末端有不同标记的成对引物,可对PCR产物进行同步纯化和鉴定。产物经生物素标记,可用连有抗生物素蛋白或共类似物的吸附剂加以分离。对异硫氰酸荧光素标记的DNA引物进行碱变性和洗脱,即可定量测定荧光标记的吸人量。为测试实用性,用2个互补于人bcr外显子n的104bp靶序列的寡聚核普酸引物,…     

用PCR技术诊断水稻的白叶枯病抗性

植物育种中应用分子标记辅助选择要求分子标记与目的基因紧密连锁,而且分析手段经济简便、重复性好。Ｘａ２１是最近发现的一个具有广谱抗性的水稻白叶枯病抗性基因,利用一个含Ｘａ２１基因的品系ＩＲＢＢ２１分别与２个感病品种杂交获得２个Ｆ_２群体。用４对引物分别对３个亲本进行ＰＣＲ分析,其中一对引物（ＰＢ７８）的ＰＣＲ产物在抗、感病品种间可揭示多态性。对２个Ｆ_２群体进一步的遗传分析表明,ＰＣＲ标记和Ｘａ２１的白叶枯病抗性紧密连锁,其重组率仅为２．４８％。根据该标记选择基因型纯合的抗性植株,其准确率可达１００％。本文还就植物育种中分子标记的检测途径进行了评价。     

HBV─C基因探针的制备及应用

应用ＰＣＲ技术扩增出ＨＢＶＤＮＡＣ基因片段并与ｐＡＴ１５３质粒重组,转化到Ｅ．ｃｏｌｉＲＲＩ中,经体内扩增,提纯,用光生物素标记,制备了Ｃ基因的重组质粒探针。该探针检测灵敏度在Ｓｏｕｔｈｅｒｎ印迹中达１ｐｇ,在点印迹中为５ｐｇ。用此探针以Ｓｏｕｔｈｅｒｎ印迹方式配合ＰＣＲ技术检测乙肝病人血清中的ＨＢＶＤＮＡ,在５３例ＰＣＲ产物电泳检测阴性的样品中,Ｓｏｕｔｈｅｒｎ杂交又检出１８例阳性。     

直接逆转录原位PCR检测实验性汉坦病毒感染乳鼠脑组织中病毒RNA及其与原位分子杂交的比较

出生后２～３ｄ的昆明种乳鼠,经腹腔接种１００个半数致死量的陈株汉坦病毒,每只０．０５ｍｌ,于接种后１、２、４、６、８、１０、１２、１４ｄ处死动物,每个时间点３～６只不等,取其脑组织固定于４％的多聚甲醛中,石蜡包埋制备５μｍ的连续组织切片,每时间点取１～２例组织切片,用逆转录原位ＰＣＲ（ＲＴ－ＩＳＰＣＲ）方法检测组织中病毒Ｓ片段ＲＮＡ,组织脱蜡后,经ＤＮａｓｅ、蛋白酶Ｋ、消化等予处理,用汉坦病毒ＲＮＡＳ片段特异的一对引物,在组织切片上进行病毒ＲＮＡ的逆转录和ＰＣＲ过程,直接将ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ掺入到扩增产物中,经过３０个ＰＣＲ循环后,用碱性磷酸酶标记的抗ｄｉｇｏｘｉｇｅｎｉｎ抗体免疫组化检测扩增产物,连续组织切片用ｄｉｇｏｘｉｇｅｎｉｎ标记的汉坦病毒Ｍ片段Ｇ２编码区ＲＴ－ＰＣＲ扩增产物的和Ｓ片段特异性探针进行原位分子杂交并与ＲＴ－ＩＳＰＣＲ结果进行比较,另外应用免疫组化检测该基因表达产物病毒核抗原（ＮＰ）,结果,ＲＴ－ＩＳＰＣＲ在病毒感染１ｄ的乳鼠脑组织中检测到病毒ＲＮＡ扩增产物,扩增产物定位于神经细胞胞浆内,而原位分子杂交和免疫组化检测阴性。在病毒感染２ｄ及２ｄ以后的乳鼠脑组织中ＲＴ－Ｉ     

水稻广亲和基因（WCG）近等基因系的随机引物PCR分析

应用随机引物ＰＣＲ（ＲａｎｄｏｍＰｒｉｍｅｒＰＣＲ）技术分别在水稻广亲和基因（ＷＣＧ）的近等基因系和色素原基因（Ｃ）的分离群体库中寻找与ＷＣＧ和Ｃ基因连锁的分子标记。对于ＷＣＧ近等基因系,在２２６个随机引物中初筛到２２个显示多态性片段的引物。根据理论值计算,在２２个多态性片段中预期有２０个与ＷＣＧ连锁。在这些连锁标记中距ＷＣＧ最近的可达０．５ｃＭ。同样在分离群体库的筛选中有１０个扩增产物与Ｃ基因连锁。     

Method for preparing single-stranded DNA templates for Pyrosequencing using vector ligation and universal biotinylated primers

In Pyrosequencing, the addition of nucleotides to a primer-template hybrid is monitored by enzymatic conversion of chemical energy into detectable light. The technique yields both qualitative and quantitative sequence information because the chemical energy is released by a stoichiometric split off of pyrophosphates from incorporated deoxynucleotide triphosphates and a defined nucleotide dispensation order is given. Because Pyrosequencing works best if single-stranded DNA templates are used, template generation usually requires PCR with a target-specific biotinylated primer and a subsequent purification involving interaction of the biotin label with immobilized streptavidin. To circumvent the need for numerous and expensive template-specific biotinylated primers, we developed a method that uses the ligation of amplified DNA fragments into a plasmid vector, thereby facilitating subsequent PCR using a universal vector-specific biotinylated primer. This approach allows easy and straightforward isolation of single-stranded templates of any PCR product. As a proof of principle, we used the method for genotyping two single-nucleotide polymorphisms in the human genes CARD15 and A2M and for characterization of four multisite variations in the human DEFB104 gene.     

An abnormally long HIV-1 env DNA PCR product due to altered sequences of primer binding sites

To investigate the generation of an abnormally long HIV-1 env PCR DNA product the latter was cloned and sequenced followed by sequence analysis of HIV-1 primer binding sites. We found that the formation of an abnormally long PCR product was due to HIV-1 env sequence alteration (a) in the reverse primer binding site resulting in faulty primer binding and (b) downstream from the forward primer sequence resulting in a new binding site with reverse complementary sequence with respect to the forward primer at the opposite end of the PCR product. Both changes led to amplification of a longer PCR product with forward primer alone. Our results indicate that the HIV-1 genetic diversity in the env gene can lead to amplification of a specific PCR product of unexpected size which can be disregarded in the absence of its cross-validation.     

基因扩增产物的固相杂交-酶联显色方法的建立

建立基于基因扩增技术的简便、快速的病毒核酸定量检测方法．将标记有生物素的寡核苷酸引物所扩增的病毒基因产物,与通过共价键结合在微孔反应板上的特异性探针进行快速杂交,然后通过辣根过氧化物酶标记的抗生物素进行酶联显色,读取光密度值．应用本方法对血清中乙型、丙型肝炎病毒核酸定量检测,灵敏度分别可达１－５拷贝／反应．此方法简便、快速、特异性好、敏感性高、半定量指标客观,可广泛应用于肝炎病毒感染的临床诊断和疗效评价．     

Fingerprinting the genetic diversity of the biotin carboxylase gene (accC) in aquatic ecosystems as a potential marker for studies of carbon dioxide assimilation in the dark

We designed and tested a set of specific primers for specific PCR amplification of the biotin carboxylase subunit gene (accC) of the Acetyl CoA carboxylase (ACCase) enzyme. The primer set yielded a PCR product of c. 460 bp that was suitable for denaturing gradient gel electrophoresis (DGGE) fingerprinting followed by direct sequencing of excised DGGE bands and sequence analysis. Optimization of PCR conditions for selective amplification was carried out with pure cultures of different bacteria and archaea, and laboratory enrichments. Next, fingerprinting comparisons were done in several aerobic and anaerobic freshwater planktonic samples. The DGGE fingerprints showed between 2 and 19 bands in the different samples, and the primer set provided specific amplification in both pure cultures and natural samples. Most of the samples had sequences grouped with bacterial accC, hypothetically related to the anaplerotic fixation of inorganic carbon. Some other samples, however, yielded accC gene sequences that clustered with Crenarchaeota and were related to the 3-hydroxypropionate/4-hydroxybutyrate cycle of autotrophic crenarchaeota. Such samples came from oligotrophic high mountain lakes and the hypolimnia of a sulfide-rich lake, where crenarchaeotal populations had been previously reported by 16S rRNA surveys. This study provided a fast tool to look for presence of accC genes in natural environments as potential marker for studies of carbon dioxide assimilation in the dark. After further refinement for better specificity against archaea, the new and novel primers could be very helpful to establish a target for crenarchaeota with implications for our understanding of archaeal carbon biogeochemistry.     

Determination method of sense sequence based on RT/PCR

When novel sequences are isolated by differential display and other methods, it seems useful to determine which is a sense sequence at an early stage before further experiments. A novel sequence, named MT-001, which shows enhanced expression in the permanent ischemic rat brain, was isolated by differential display. Based on this sequence, a primer set for both direction was designed. Each primer was used to make a cDNA and PCRs performed with each cDNA and both primers. One primer used in the RT step produced a PCR product at the expected position, but another primer in the reverse direction could not. This result indicated that the primer that made the expected PCR product is antisense.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Development of a simplified polymerase chain reactionenzyme immunoassay for the detection of Chlamydia pneumoniae

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae -specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays

DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon. To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed.