A rapid protocol for the authentication of isolated differential display RT-PCR CDNAs.

The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies.     

Practical strategy for identification of single nucleotide polymorphisms in fruiting mei (Prunus mume Sieb. et zucc.) from amplified fragment length polymorphism fragments

Single nucleotide polymorphism (SNP) is the most abundant form of genetic variation among individuals within a species. SNPs can be used as markers for gene discovery and for assessment of diversity. We established a practical strategy for identification of SNPs in fruiting mei (Prunus mume Sieb. et Zucc.) from amplified fragment length polymorphism (AFLP) fragments. The main modification of this procedure was optimization of the reamplification of bands excised from an AFLP gel by using a single enzyme (EcoRI) in digestion reaction to generate large AFLP fragments and to lower the number of bands on gels, using lower-concentration polyacrylamide gels (4%) and loading each sample into 4 continuous lanes, using a newly modified protocol for purification of AFLP bands from the gel, and using additional cycles for reamplification of AFLP bands. In this study, 15 groups of bands with identical migration distances from 10 fruiting mei cultivars were selected for purification. Eighty-one of the 150 chosen bands were successfully reamplified, and 67 of these reamplified polymerase chain reaction products yielded reliable sequences belonging to 13 groups. The alignment of 13 group sequences yielded 95 SNPs, for a total of 5252 bp. Among these SNPs, 73 were heterozygous in the loci of some individual cultivars. The SNP distribution was 58% transition, 40% transversion, and 2% indels. There was also 1 dinucleotide polymorphism and 1 tetranucleotide deletion.     

荧光标记mRNA差异显示技术

目的：应用荧光标记的ｍＢＮＡ差异显示技术。方法：提取未经过／经过ＩＦＮ－ＬＰＳ处理的三组人单核细胞系Ｕ９３７的总ＲＮＡ并以此为模板,采用荧光标记的锚定引物,通过逆转录、差异显示ＰＣＲ反应,经５．６％变性聚丙烯酰胺凝胶电泳分离差异条带,回收后将其再扩增。结果：三组样本的ＤＤ－ＰＣＲ产物电泳显示长３００ｂｐ￣２．０ｋｂ不等的扩增片段,条带清晰、明亮,背景低,各样本相互间的差异不仅呈有无的变化,亦表现出     

Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.

DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product.     

The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display

In many applications, an understanding of differentially expressed genes in different tissues, or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential, display [DDR-PCR] has shown, that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.     

家蝇幼虫差异表达基因的克隆筛选与分析

目的是利用mRNA差异显示(DDRT-PCR)技术对诱导后家蝇幼虫差异表达基因进行克隆分析。取诱导和未诱导家蝇(Musca domestica vicina)三龄幼虫总RNA进行mRNA差异显示反应,产物经6％非变性聚丙烯酰胺凝胶电泳展开,银染分析后,回收差异显示条带。随机选取4条诱导家蝇三龄幼虫中上调表达的基因条带,进行Northern杂交验证,有2条被验证为真实带,命名为YD1和YD2。对这2条基因片段进行T-A克隆和测序,长度分别为495bp和265bp,登录NCBI运用Blastn程序进行同源性比较,发现两者与任何已知基因同源性都低于70％,提示为两个未报道的新基因,在家蝇免疫反应中可能起着一定的作用。     

Insertion of a reamplification round into the ISSR-PCR protocol gives new flax fingerprinting patterns

We expanded the basic ISSR-PCR protocol by an additional PCR reamplification round in order to detect whether increased PCR productivity would give new bands in ISSR patterns. We found that the reamplification step had a prominent impact on the quality of the inter-simple-sequence repeat (ISSR) PCR patterns of flax, depending on the particular primer used for PCR amplification. We could clearly distinguish between two types of reamplification effect. Most ISSR primers (16 out of 21) gave no reamplification effect as usual, but five primers (23.8%) provided a new ISSR fingerprinting pattern after the 2nd reamplification round, leaving the previous 1st round pattern completely blank. Therefore, we recommend the expansion of a basic ISSR-PCR protocol for another reamplification round in order to mine out full the fingerprinting potential from ISSR-PCR method.     

正交法整体优化差异显示反应体系

mRNA 差异显示PCR( mRNA differential display PCR,DDRT-PCR)是分离差异表达基因的有效方法,但该方法的准确性极易受到外部因素和内部因素的影响。本研究采用正交法优化DDRT-PCR反应条件,充分考虑到模板浓度、锚定引物浓度、随机引物浓度、dNTPs浓度、镁离子浓度以及Taq酶用量等因素在差异显示反应过程中的交互作用,一次PCR反应即可确定最佳反应组合。将筛选出的条件用于DDRT-PCR,得到差显结果假阳性率低,重复性和稳定性好,而且简化了反应条件的优选程序,这表明正交法是优化差异显示反应条件的理想方法。为了进一步简化整个差异显示反应系统的操作程序,降低假阳性率,研究采用了非变性聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和银染显示差异带的方法,并用反向Northern法来验证回收条带,从而更加优化了差异显示反应体系。     

Recovering and reamplifying of the differentially expressed cDNA bands isolated from mRNA differential display

Methods for retrieving and reamplifying the differentially expressed cDNA bands have been modified. Direct reamplification of differentially expressed bands after cutting from a polyacrylamide gel (PAG) followed by a simple rinse and crush step has proved to be more convenient and effective than the traditional glycogen-precipitation method. Combination of 30 cycles of differential display (DD) polymerase chain reaction (PCR) and 20 cycles of standard PCR reaction also yielded higher reamplification rates.     

Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-microarray

Infections with mycobacteria are an important issue in public health care. Here we present a "proof-of-principle" concept for the identification of 37 different Mycobacterium species using 5' exonuclease real-time PCR and DNA microarray based on the region upstream of the 65 kDa heat shock protein. With our two PCR probes, one complementary to all mycobacteria species, the other specific for the M. tbc-complex, 34 species were properly classified by real-time PCR. After reamplification and hybridization to a DNA microarray, all species showed a specific pattern. All 10 blindly tested positive cultures revealed a positive real-time PCR signal with the genus probe. After reamplification and hybridization, six samples could unambiguously be identified. One sample showed a mixture of presumably three species-specific patterns and sequencing the 16S rRNA confirmed the presence of a mixture. The hybridization results of three specimens could not be interpreted because the signal to background ratio was not sufficient. Two samples considered as negative controls (LAL Reagent Water (Cambrex) and DNA of Candida albicans) gave neither a genus nor a M. tbc-complex positive PCR signal. Based on these results we consider our method to be a promising tool for the rapid identification of different mycobacteria species, with the advantage of possible identification of mixed infections or contaminations.     

The effect of DNA reamplification on RAPD-PCR results

The results of reamplification of flax DNA with random primers are represented. It was shown that eight of 17 decameric primers used in the experiments had an reamplification effect: the intensity of the bands increased; new bands and/or new polymorphic bands appeared in the gel sample. The results confirm the effectiveness and necessity of reamplification in case of uncertain or ambiguous interpretation of RAPD analysis making possible to select the highly-informative primers.     

Expression of stage-specific genes during zygotic gene activation in preimplantation mouse embryos

The expression of mouse two-cell stage specific genes was studied using the modified DDRT-PCR method, which overcame the paucity of the experimental materials of preimplantation embryos. Embryo tissues equivalent to that of four blastomeres are sufficient for amplification of target genes as visualized using polyacrylamide gel. Sequence analyses and reverse Northern blots indicate that the genes of ATPase 6 and Ywhaz are expressed specifically in two-cell embryos. ATPase 6 is essential for one-cell to two-cell transition and plays an important role in establishment of oxidative phosphorylation, while Ywhaz is related to initiating cellular communication system.     

差异显示反转录PCR技术研究进展

分析一对细胞或组织在不同状态下基因表达的差异,已成为分子生物学研究领域的热点之一.近年来,用于识别差异表达基因的方法已发展起来多种.DDRT-PCR是近年来较为广泛应用的一种技术.理论上,DDRT-PCR技术比较简单,但实施起来却存在着假阳性率高,凝胶中单条cDNA带成分不均一, 所获cDNA仅代表着mRNA 3′UT区(约300 bp)以及一些低拷贝数mRNA不能有效被呈现等问题.对DDRT-PCR技术的改良也主要集中在解决这些问题方面.     

东亚砂藓取材部位对提取其总RNA及DDRT-PCR的影响

分别采用新鲜东亚砂藓植物体先端、基部及中部作为材料,采用改良的SDS法,提取及纯化三部分的总RNA。比较RNA产率、纯度及分析电泳图谱来确定适于东亚砂藓RNA分离的最佳部位。并运用mRNA差异显示方法比较了东亚砂藓不同部位的差异。实验结果显示,东亚砂藓植物体的先端适于其总RNA提取及mRNA差异显示的研究。     

Coincidence painting: a rapid method for cloning region specific DNA sequences.

We have developed a novel coincidence cloning strategy, termed Coincidence Painting, which enables the rapid generation of large numbers of region specific sequences. Coincidence Painting utilises Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification of flow sorted derivative translocation chromosomes. The PCR products are hybridised in situ onto specific flow sorted chromosomes for coincident sequence selection. Eluted and reamplified material is then cloned using a novel insert end revelation and ligation technique. Cloned inserts range in size from 150-1300 bps of which approximately 54% appear to be single copy sequences. The cloning method permits the excision of vector free probe for library hybridisation screening and the small insert size facilitates analysis for the generation of sequence tagged sites (STSs). We have used such clones successfully for YAC screening by PCR and for cosmid screening by filter hybridisation. This new methodology should allow the rapid saturation with probes of regions defined by specific translocation breakpoints.     

红莲优6号杂交水稻与亲本三系不同发育时期幼苗叶片基因表达差异分析

运用DDRT-PCR对红莲型杂交稻组合红莲优6号及其亲本、保持系（粤泰A、粤泰B、9311）的一叶期、三叶期叶片基因表达状况进行分析。结果表明：杂种与亲本之间在同一发育时期基因表达既有质量上又有数量上的差异,但差异表达基因所占比较较小;而对于同一材料,一叶期、三叶期的叶片cDNA扩增带型相似,没有发现基因质上的差异表达,说明一叶期与三叶期幼苗叶片基因表达差异很小。实验也证明DDRT-PCR技术结合银染方法是一种简单快速分析差异表达基因的有效方法。