Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification.

Two simple methods for site-specific mutagenesis are described and compared. In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product. In the first method, the two products are combined, denatured and reannealed prior to transformation of E. coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E. coli without prior manipulation, resulting in transformation of E. coli with the recombinant of interest by recombination in vivo. Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified. This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification. In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors.     

PCR clamping

An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.     

虚拟细胞

虚拟细胞是20世纪末在国外刚刚兴起的一种细胞生物学研究方法,主要是通过计算机建立人工细胞模型,模拟细胞内外环境,从而进行生物学的研究和探索。虚拟细胞包含了多门学科前沿的研究成果,具有十分重要的意义。现从其发展历程、应用及其对生物学工作者的关系等方面进行综述。     

Virtual conferences

An interview with Barry Hardy, conducted in May and June 1996 by e-mail and on 12 June 1996 at a MOO site. The development of the Internet has provided the opportunity o f conducting meetings and conferences electronically. In this interview, Barry Hardy outlines the main principles involved in staging and attending a 'virtual meeting', and issues an invitation to the readers o f trends in CELL BIOLOGY to become involved in organizing the first international electronic cell-biology conference.     

Virtual neanderthals

Our closest hominid relatives may have died out 30,000 years before the arrival of the computer, but thanks to modern genomics and scanning technology, they are now very present in the 21st century and can even help us understand our own species.     

卫氏并殖吸虫PCR和实时荧光PCR快速检测方法的建立

目的建立快速、灵敏、特异的分子生物学检测卫氏并殖吸虫的方法。方法根据卫氏并殖吸虫的特异性基因序列,设计适合于PCR检测的特异性引物及实时荧光PCR特异性引物和探针,并进行灵敏性和特异性试验。结果设计的引物和探针特异性强,所建立的检测方法灵敏度高。应用实时荧光PCR方法的检测灵敏度可达到0.1 pg/μL,比PCR方法的灵敏度高三个数量级。结论本研究所建立的PCR和实时荧光PCR技术检测卫氏并殖吸虫方法的特异性强,灵敏度高,为卫氏并殖吸虫感染的诊治提供了快速的检测技术手段。     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

Primer design versus PCR bias in methylation independent PCR amplifications

Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten “CpG-free” primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the “CpG-free” primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1–0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.     

The PCR suite

The web application PCR Suite is an extension of the primer design program Primer3. It allows the design of primer sets encompassing single nucleotide polymorphisms, all exons of a single gene, all open reading frames in a list of cDNAs or the creation of overlapping PCR products.     

High-throughput droplet PCR

The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.     

限制性酶切片段差异显示及其应用

差异显示技术(DD)-PCR是一种研究基因表达差异的重要而应用广泛的方法,传统的差异显示法由于在PCR时采用Poly（T）引物和随机引物而导致较高的假阳性率和产物的近Poly(A)非编码区的大量扩增。改进后的限制性酶切片段差异显示技术(RFDD)-PCR采用ToqI酶切双链cDNA,连上特殊设计的接头,再用经特殊设计的特异性配对于接头的引物来扩增,因此能重点扩增编码区并能极大地消除假阳性率。由于扩增时引物就带有荧光或放射性核素标记,还使得差异显示条带的检测更为方便、灵敏。本简要介绍了该法的原理、步骤、应用及其优缺点。     

Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.