Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.     

Toward metrological traceability for DNA fragment ratios in GM quantification. 3. Suitability of DNA calibrants studied with a MON 810 corn model

The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio.     

Fate of Maize DNA During Steeping,Wet-Milling,and Processing

The fate of DNA during steeping, wet-milling, and subsequent processing of maize was examined using a sensitive polymerase chain reaction (PCR-based) detection system. The system used specific amplification of maize DNA sequences by primers generated toward plant nuclear- and chloroplast-encoded genes. The PCR method facilitated analysis of DNA content in food products, which is an important issue in use of genetically modified organisms. In a conventional laboratory wet-milling countercurrent steep system, DNA was detected in maize kernels throughout the process but was not found in steepwater. After kernels were wet-milled, DNA was detected in the starch, germ, coarse fiber, and wet gluten fractions but not in the fine fiber fraction. When dried by heating at 135°C for 2 hr, DNA was degraded to undetectable levels in the wet-milled gluten fraction and hydrated kernels. DNA was not detected in feed pellets, starch, dextrose, sorbitol, or high-fructose maize syrup made from industrial wet-milled samples. Although DNA could be detected in laboratory wet-milled fractions, some degree of degradation occurred after extended exposure to steepwater. Countercurrent steepwater samples from the later stages of the steeping process were able to degrade DNA. The level of DNA degradation appeared to correspond to the presence of sulfur dioxide and may represent a physiochemical rather than an enzyme-mediated process. Our results indicate that some steps in the steeping and wet-milling process can degrade maize genomic and plastid DNA.     

An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR

Background, aim and scope  An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg²⁺ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Materials and methods  Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex ^TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. Results and discussion  The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:10³. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™ spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. Conclusions  A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. Recommendations and perspectives  This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.     

DNA identification of commercial ginseng samples

An investigation was performed with the objective of developing a DNA-based protocol for the identification of commercial samples of the herbal compound ginseng. There are currently two major herbal products referred to as ginseng. They are Korean or Chinese ginseng (Panax ginseng) and American ginseng (Panax quinquefolius). The market for ginseng in the United States is estimated to be approximately $300 million annually. Current tests for ginseng species identification rely on expert botanical identification of fresh plant/root specimens or on biochemical characterization of active and marker compounds (e.g., ginsenosides). For the determination of the feasibility of ginseng identification by DNA analysis, a strategy based on the direct DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region was developed. Other genetic tests included sequence analysis of the chloroplast ribulose 1,5-bisphosphate carboxylase large subunit gene and DNA fingerprinting by the rapid amplification of polymorphic DNA technique. To confirm the results, each ginseng sample was identified using high-performance liquid chromatography. All methods were successful in distinguishing American from Korean ginseng. In addition, the protocol was improved for the isolation of genomic and plastid DNA from commercial ginseng preparations by incorporating an impact homogenization step into the standard column chromatography purification procedure.     

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.     

西北地区耕地土壤真菌DNA提取方法比较

由于西北土壤理化性质的复杂性和真菌特殊性,所以从土壤中提取真菌基因组DNA就相对细菌更困难。在2种常用的土壤微生物基因组DNA提取方法与在传统提取方法的基础上,结合了一种专门适用于真菌的提取方法进行了比较,并且利用真菌28SrDNA通用引物U1/U2进行扩增。三种提取方法比较结果表明:SDS法提取的DNA纯度最低,传统CTAB-SDS的DNA产量最低,实验室的提取方法既可以提高DNA产量又可以保证DNA的片段完整性,并且本实验室的提取方法扩增效果最好,可广泛应用于西北地区土壤真菌的分子生物学研究。     

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).     

Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.     

DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.     

Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005 ng of rice genomic DNA, respectively. To test the practical use of this SPS gene as an endogenous reference gene, we have also quantified the beta-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPS gene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.     

Comparison of three DNA extraction methods for feed products and four amplification methods for the 5'-junction fragment of Roundup Ready soybean

Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients.     

Validated method for quantification of genetically modified organisms in samples of maize flour

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.     

A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples

Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.     

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.     

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。     

干制羊肉基因组DNA不同提取方法的比较

为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL^-1,A₂₆₀/A₂₈₀值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。     

Comparison of several methods for the extraction of DNA from potatoes and potato-derived products

Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods.     

紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法.