Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

HPLC purification of polymerase chain reaction products for direct sequencing

Same day PCR amplification and sequencing is desired in situations where one needs to sequence a number of PCR products. The rapid, high-yield purification of PCR products via the use of high performance, anion-exchange chromatography yields sequencing results comparable to those obtained from techniques requiring subcloning of the PCR product. This can be achieved by standard dideoxynucleotide sequencing technology without the need to prepare prelabeled primers and additional internal primers or to gel purify the PCR product. In addition, this chromatographic technique offers the potential of isolating several PCR products from the same amplification mixture.     

Rapid generation of random mutant libraries

A simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification is described. This will be especially useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Here we investigate various parameters to optimize this approach and we demonstrate that as little as 1 pmole of PCR fragment can generate a library with greater than 104 clones in a single transformation without ligation.     

扩增内标及其在食源性致病菌PCR检测中的应用

虽然PCR技术不断地得到了发展和改进,但检测结果容易出现假阴性而影响检测准确性的现象一直没有得到很好的解决。现在大多数学者普遍认为,在PCR体系中加入扩增内标(即一段人工构建合成的DNA序列或者是一段致病菌的看家基因序列)能有效指示假阴性现象的出现,是PCR检测技术标准化的措施之一。本文将从PCR检测方法中假阴性出现的原因、扩增内标的构建以及扩增内标在PCR检测方面的应用三方面进行综合评述,并结合本实验室的工作基础,介绍扩增内标的简捷构建过程和应用要点,希望在不影响检测灵敏度的前提下,发挥扩增内标对假阴性的指示作用。     

二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用

本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行ＰＣＲ扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记ＰＣＲ产物,简便、快速,分辨率可达１ｂｐ,并可用多对引物同时进行多重ＰＣＲ分析。用此方法对ＤＭＤ家系成员ｄｙｓｔｒｏｐｈｉｎ基因的５＇－脑型外显子止游区和３＇－非翻译区的两个（ＣＡ）。位点进行了扩增片段长度多态性分     

The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins

A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.     

Solid-phase plate-reader quantification of specific PCR products by measurement of band-specific ethidium bromide fluorescence

Real-time PCR is widely employed to quantify PCR products across a range of applications. However, accurate real-time PCR is not always technically feasible, and alternative methods for PCR product quantification can be expensive and time consuming to validate. We have developed an inexpensive, rapid, and immediately accessible protocol to quantify PCR products, by measuring ethidium bromide fluorescence of PCR products excised from agarose gels. This protocol has relevance to a broad range of methods in molecular biology where quantification of PCR products is necessary.     

Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells

DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers to increase the specificity and sensitivity of amplification. Breaksites are then mapped by batch sequencing LM-PCR products. This allows easy identification of multiple breaksites per reaction without tedious fractionation of PCR products by gel electrophoresis or cloning. Breaksite batch mapping requires little starting material and can be used to identify either single- or double-strand breaks.     

Fusion PCR and gene targeting in Aspergillus nidulans

We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.     

Rapid and Efficient Gene Splicing Using Megaprimer-Based Protocol

Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Microsatellite analysis using a two-step procedure for fluorescence labeling of PCR products.

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classical PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the product on only one strand. The products can be applied without prior purification directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals those of the classical 32P or fluorescent primer labeling methods, and the method is definitely less costly. Since the interpretation of the results is easier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mapping projects requiring analysis of a large number of microsatellites.     

A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs

We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products. The method is suitable for use with a proof-reading polymerase such as pfu, or any other heat-stable polymerase which produces a blunt-end product. The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%. Because the sequences of the sticky ends are defined by the user and potentially can be of any length, the method can also be exploited for the one-step construction of recombinant plasmids from multiple functional cassettes, without the use of restriction enzymes.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient

With the advent of the highly active antiretroviral therapy (HAART), the natural course of HIV infection has markedly changed and opportunistic infections including toxoplasmosis have declined and modified in presentation, outcome and incidence. However, TE is a major cause of morbidity and mortality especially in resource-poor settings but also a common neurological complication in some countries despite the availability of HAART and effective prophylaxis. In most cases toxoplasmosis occurs in brain and toxoplasmic encephalitis (TE) is the most common presentation of toxoplasmosis in immunocompromised patients with or without AIDS. The need of a definitive diagnosis is substantial because other brain diseases could share similar findings. Rapid and specific diagnosis is thus crucial as early treatment may improve the clinical outcome. Classical serological diagnosis is often inconclusive as immunodeficient individuals fail to produce significant titres of specific antibodies. Polymerase chain reaction (PCR) has a high diagnostic value in the acute disease, but like many 'in-house' PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols. This article summarises the clinical manifestations, diagnostic procedures and management strategies for this condition.     

A simple and fast method for cloning and analyzing polymerase chain reaction products

A method describing a fast and efficient way for cloning polymerase chain reaction (PCR) products is presented that involves end repair and purification of the PCR product, followed by kinasing and ligation to the vector with the use of a temperature gradient. Efficiency of ligation was estimated to be 50%–70%. Following transformation, cells are plated on MacConkey agar. Bacteria from selected colonies are used directly from the plates for screening without any subsequent purification. Using this protocol, PCR products can be efficiently cloned quickly and economically.     

Development of a synthetic positive control which also detects plasmid contamination in diagnostic polymerase chain reaction

This paper describes a technique for the development of a positive control for use in a nested PCR to show that the PCR has worked correctly with both outer and inner primers designed for diagnostic amplification of 618 bp and 317 bp products respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: 1) making it visually easy to detect plasmid contamination and thus, prevent false positives from the plasmid; 2) develop a positive control when the target organism is at a very low prevalence so initial detection is not relied on for cloning positive controls. This will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives; or 3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrates how to use large oligonucleotide primers in PCR without non-specific binding occurring.     

Development of a Synthetic Positive Control Which Also Detects Plasmid Contamination in Diagnostic Polymerase Chain Reaction

This paper describes a technique for developing a positive control for use in a nested PCR to show that the PCR has functioned correctly with both outer and inner primers designed for the diagnostic amplification of 618 and 317 bp products, respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: (1) making it visually easy to detect plasmid contamination and thus prevent false positives from the plasmid; (2) develop a positive control when the target organism is at a very low prevalence, so initial detection is not relied on for cloning positive controls (this will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives); or (3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrate how to use large oligonucleotide primers in PCR without nonspecific binding occurring.