Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

DNA elution and amplification by polymerase chain reaction from dried blood spots.

A quick, sensitive and easily automatizable method for PCR amplification of genomic DNA eluted from dried blood spots is described. DNA is eluted from a 3-mm spot routinely used for neonatal screening of inherited diseases either by boiling or by sonication. A preliminary and brief spot-autoclaving step is mandatory to ensure optimal and reproducible PCR amplifications. Only 1% of the eluted DNA is required for PCR analysis allowing the execution of multiple genetic tests on the same blood spot. The method has been successfully applied to the detection of a known phenylketonuria-causing mutation and will facilitate the analysis of the genetic repository provided by Guthrie's cards stored in neonatal screening laboratories.     

The polymerase chain reaction: an improved method for the analysis of nucleic acids

Summary The polymerase chain reaction (PCR) is a method for the selective amplification of DNA or RNA segments of up to 2 kilobasepairs (kb) or more in length. Synthetic oligonucleotides flanking sequences of interest are used in repeated cycles of enzymatic primer extension in opposite and overlapping directions. The essential steps in each cycle are thermal denaturation of double-stranded target molecules, primer annealing to both strands and enzymatic synthesis of DNA. The use of the heat-stable DNA polymerase from the archebacterium Thermus aquaticus (Taq polymerase) makes the reaction amenable to automation. Since both strands of a given DNA segment are used as templates, the number of target sequences increases exponentially. The reaction is simple, fast and extremely sensitive. The DNA or RNA content of a single cell is sufficient to detect a specific sequence. This method greatly facilitates the diagnosis of mutations or sequence polymorhisms of various types in human genetics, and the detection of pathogenic components and conditions in the context of clinical research and diagnostics; it is also useful in simplifying complex analytical or synthetic protocols in basic molecular biology. This article describes the principles of the reaction and discusses the applications in different areas of biomedical research.     

从土壤中提取细菌和芽孢核酸用于PCR的研究

本文探讨了土壤中提了细菌及其芽孢DNA用于PCR扩增检测的方法,我们采用的碘化钠裂解,玻璃粉吸附方法,可以有效提取和纯化土壤中的细菌及芽孢的核酸,操作简便,不需要特殊设备,提取的核酸可直 用于PCR扩增反应,我们用炭疽芽孢杆菌A16R疫苗株的芽孢的类鼻疽伯克霍尔德氏菌污染土壤,使用这一方法从中提取核酸,并用于PCR扩增,证实了该方法的实用性。     

Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification.

In this report we describe a simple and rapid technique using DNA affinity columns that permits direct extraction of bacterial plasmids from a variety of foods for polymerase chain reaction amplification. The procedure was used to detect virulent enteroinvasive Escherichia coli in several artificially seeded matrices, including seafoods, greens, dairy products, enrichment media, and water. Polymerase inhibitors present in both foods and enrichment media were removed efficiently.     

Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification.

In this report we describe a simple and rapid technique using DNA affinity columns that permits direct extraction of bacterial plasmids from a variety of foods for polymerase chain reaction amplification. The procedure was used to detect virulent enteroinvasive Escherichia coli in several artificially seeded matrices, including seafoods, greens, dairy products, enrichment media, and water. Polymerase inhibitors present in both foods and enrichment media were removed efficiently.     

Isolation of eicosapentaenoic acid-producing fungi from soil based on polymerase chain reaction amplification

A method was developed for rapid screening and isolation of eicosapentaenoic acid (EPA)-producing soil fungi through polymerase chain reaction (PCR) amplification. Genes coding for delta6 fatty acid desaturase and delta5 fatty acid desaturase were used as molecular markers for screening these EPA-producing fungi from soil. Three out of 65 soil fungi gave positive results through PCR amplification. Two out of these three strains were found to produce EPA when they had grown in 80 ml potato/dextrose liquid medium at (25 +/- 1) degrees C for 144 h. The EPA yields were 215.81 mg 1(-1) and 263.80 mg 1(-1), respectively. The other positive strain was detected to produce arachidonic acid (AA). This study indicates that molecular detection of genes encoding delta6 and delta5 desaturases is an efficient method for primary screening of EPA- or its related polyunsaturated fatty acids (PuFAs)-producing fungi, which can improve the screening efficiency prominently.     

Characterization of polymerase chain reaction amplification of specific alleles

Under certain conditions, polymerase chain reaction (PCR) can be used to differentially amplify one allele over another. To characterize the phenomenon, we have made a series of PCR primers and determined whether differential amplification could be detected after agarose gel electrophoresis. Two allele pairs were examined; one pair represents a transversion and one pair represents a transition. The following conclusions emerge: (i) when the 3' or the 3' penultimate base of the oligonucleotide mismatched an allele, no amplification product could be detected; (ii) when the mismatches were 3 and 4 bases from the 3' end of the primer, differential amplification was still observed, but only at certain concentrations of magnesium chloride; (iii) the mismatched allele can be detected in the presence of a 40-fold excess of the matched allele; (iv) primers as short as 13 nucleotides were effective; and (v) the specificity of the amplification could be overwhelmed by greatly increasing the concentration of target DNA.     

Direct amplification of unknown genes and fragments by Uneven polymerase chain reaction

Gene cloning is a time-consuming task for molecular biologists, because it often takes weeks or months to construct, screen and finally clone a gene from a DNA library. Thus, more effective methods are needed for gene cloning. This paper describes a modified polymerase chain reaction (PCR) cycling condition, Uneven PCR, to generate specific unknown fragments or genes directly from total DNA instead of cloning fragments from DNA libraries. The essence of the method is to use two different annealing temperatures in consecutive PCR cycles to effectively amplify the target products while inhibiting the synthesis of non-specific products. Under favorable conditions, a desired DNA fragment or gene in the size range up to approximately 4 kb can be obtained and ready for cloning within a day or two.     

Rapid polymerase chain reaction amplification using intact bacterial cells

We have demonstrated that efficient polymerase chain reaction amplifications from chromosomal DNA can be carried out using whole bacterial cells as the starting material. Cells from the liquid or solid cultures can be used directly, without any pre-treatment, thus eliminating the need for DNA isolation.     

A comparison of methods for DNA extraction from paraffin-embedded tissue for microsatellite instability analysis by polymerase chain reaction

In a prospective study, nuclear DNA was extracted from colorectal tumours and normal mucosa which had been fixed in buffered formalin and embedded into paraffin. DNA-extraction was performed using three different methods: a commercial kit which was not especially created for this use; a known fast procedure without DNA-cleaning steps; and a more conventional DNA-preparation protocol with DNA-cleaning. Using the polymerase chain reaction (PCR), DNA was amplified by being targeted onto two β-globin fragments with different lengths (536 bp and 989 bp) and (CA)n repeats localized on chromosome 5q (D5S346) and chromosome 17p (TP53CA) with a length of about 100 bp for detection of microsatellite instability. The success rate of microsatellite amplification was 100% with all methods. The 536 bp β-globin fragment could be amplified with a success rate ranging from 40% to 100%. The amplification of the 989 bp β-globin fragment was unsuccessful. Significant differences were observed between the three methods in the final DNA concentration and DNA yield. In microsatellite instability studies of paraffin-embedded tissues, the investigator can expect a high success rate of nearly 100% using any of the described methods.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.