PrimRglo: a multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection

We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.     

Strand specific PCR amplification of low copy number DNA.

A method for the amplification of a single DNA strand at low copy number is described. It is a wholly PCR based approach which involves an initial linear amplification of the target using a tagged strand specific primer. This is followed by classical PCR amplification of the progeny using a pair of primers, one specific for the sequence tagged onto the 5' end of the first round primer, the second specific for the target sequence. Given the protocol used the ratio of the two strands in the final amplification product was 50:1.     

甘蓝型油菜PEP基因片段的克隆和种子特异性反义表达载体的构建及转基因油菜的获得

丙酮酸羧化酶(PEP)是控制油菜蛋白质/油脂含量比例的一个种子的含油量.本研究利用PCR法从甘蓝型油菜花油5号(H045)克隆了PEP基因片段,并与载体pBI121-B构建了反义PEP基因的种子特异性植物表达载体,通过激光微束穿刺法将其转化到甘蓝型油菜中,目前已获得了转基因植株.     

一种新的用于提高lea3基因扩增特异性和产量的增强剂（英文）

在对目的DNA序列尤其是高GC含量的片段进行PCR扩增的时候,经常需要对一些试验条件进行优化。在一定条件下,二甲基亚砜、甲酰胺、甘油、NP-40和Tween20等可以在某种程度上提高PCR的特异性和效率。我们在对禾本科lea3基因进行分离克隆时发现了一种新的可以提高PCR产量和特异性的物质——极高热稳定单链结合蛋白（ETSSB）,研究发现在每50μlPCR反应体系中加入200ng的ETSSB,可以有效地抑制DNA片段的非特异性条带的产生,并可以提高目的片段的产量。     

Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid

We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.     

Tagged polymerase chain reaction subtractive hybridization for the enrichment of phage display random peptide libraries

Affinity selection of phage display peptide libraries is routinely used for isolating peptides capable of binding a range of molecules, including antibodies and receptors. This process is most successful when the selecting molecule is relatively pure, for example, a monoclonal antibody. However, isolation of peptides able to bind to target molecules present in a complex mixture is more difficult because the affinity selection process isolates peptides capable of binding to all molecules present in the mixture. Here we describe the development of a tagged polymerase chain reaction (PCR) subtractive hybridization method that is universally applicable for the targeted isolation of peptides able to bind to unique molecules within a complex mixture. We also describe a discriminatory limiting dilution PCR method that can be used to optimize hybridization conditions.     

Combination of amplification and post-amplification strategies to improve optical DNA sensing

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.     

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5′- and 3′-regions of a target gene. Of the four primers used in amplification of the 5′- and 3′-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5′- or 3′-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5′ and 3′ PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.     

Determination of the sequence coding for the beta subunit of the human high-affinity IgE receptor.

The cDNA encoding the beta subunit of the human high-affinity IgE receptor was cloned by a combination of various polymerase chain reactions (PCR). A major portion of the beta cDNA was amplified using primers homologous within the sequences of rat and mouse. The 3' unknown sequence was preferentially amplified using the RNA template-specific PCR and the improved two-step PCR. The 5' unknown sequence was specifically amplified by our newly developed PCR walking. Random heptanucleotides tagged with a unique sequence at the 5' end were used as the walking primer. Finally, the entire coding region was amplified and sequenced. The two extracellular loops of the human beta subunit were the least homologous to those of rat and mouse.     

Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.