scispace - formally typeset
Search or ask a question
Journal ArticleDOI

A rapid DNA isolation procedure for small quantities of fresh leaf tissue

01 Jan 1980-Phytochemistry (The Phytochemical Section of the Botanical Society of America)-Vol. 19, Iss: 1, pp 11-13
TL;DR: From the kinetic data, it becomes evident that the reductive amination reaction is highly adaptive to the ammonium environment.
About: This article is published in Phytochemistry.The article was published on 1980-01-01. It has received 14480 citations till now. The article focuses on the topics: Ammonium & Substrate (chemistry).
Citations
More filters
Journal ArticleDOI
04 May 2011-PLOS ONE
TL;DR: A procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs) is reported, which is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches.
Abstract: Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

5,163 citations


Cites methods from "A rapid DNA isolation procedure for..."

  • .... The 43 barley lines were selected from the larger set of 83 OWB lines to maximize recombination. High molecular weight DNAs were extracted from leaves of single plants using a standard CTAB protocol...

    [...]

Journal ArticleDOI
TL;DR: This is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage.
Abstract: A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.

1,869 citations

Journal ArticleDOI
TL;DR: Nine newly explored regions of the chloroplast genome offer levels of variation better than the best regions identified in an earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.
Abstract: Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

1,840 citations

Journal ArticleDOI
TL;DR: The results of this study show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.
Abstract: Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.

1,763 citations

Journal ArticleDOI
28 Feb 2012-PLOS ONE
TL;DR: The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence.
Abstract: Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.

1,492 citations

References
More filters
Journal ArticleDOI
TL;DR: There is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.

510 citations

Journal ArticleDOI
TL;DR: It is suggested that sharp discontinuities in Lineweaver-Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair-Koshland model, but not by the model of Monod, Wyman & Changeux.
Abstract: 1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD+ and NADP+ and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis–Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7·0, Lineweaver–Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver–Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair–Koshland model, but not by the model of Monod, Wyman & Changeux.

139 citations

Journal ArticleDOI
TL;DR: The partially purified glutamic dehydrogenase from corn leaves has a pH optimum of 8.1, a coenzyme specificity for DPN, and a substrate specificity for l -glutamate, and was not inhibited by cyanide, hydroxy acids, or o-iodosobenzoate.

100 citations

Journal ArticleDOI
TL;DR: The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer has a total of I8 nucleotide binding sites, and glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient.
Abstract: The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer (composed of six identical polypeptide chains) has a total of I8 nucleotide binding sites. They can be subdivided into three classes. A first set of six binding sites, the active sites, is occupied by NAD(P)H with nearly the same affinity for both coenzymes, and accompanied by negative cooperativity between the binding sites within the oligomer. The second set of six binding sites, the nonactive sites, binds the coenzyme less tightly than the first set and ADP competes with NAD(P)H for these sites. The third set of six binding sites, the GTP sites, is responsible for the GTP binding. ADP abolishes the negative cooperativity of the NAD(P)H binding to the active sites and causes a weaker binding of both reduced coenzymes to these sites (KE. ADP, NAD(P)H = 70 pM for NADH and 64 pM for NADPH). Also the reduced coenzymes weaken the binding of ADP to the enzyme. The dissociation constant for the enzyme * ADP complex is found to be 17 pM in the presence and 3 pM in the absence of NAD(P)H. The results are interpreted on the basis of free energy conservation in internal equilibria. The coupling energy AE”, was calculated to be about I kcal/mol. In contrast to ADP/NAD(P)H, GTP and NAD(P)H enhance each other’s binding to glutamate dehydrogenase causing positive cooperativity which is stronger in the case of NADH than of NADPH. The Hill coefficient is found to be 2.1 for NADH and 1.8 for NADPH. The nonactive sites are occupied by NADH in the presence of GTP with a dissociation constant of 23 $5 indicating a three-fold enhancement of the affinity of the enzyme for NADH in the presence of GTP. This is not the case with NADPH, where the dissociation constant is about 600 pM. This value is similar to the dissociation constant of the binary complex. The influence of glutamate on the enzyme * NAD(P)H binding is similar to the effect obtained in the presence of GTP; glutamate and GTP, however, are most likely bound to Merent sites. Glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient of 2.4 for both reduced coenzymes. The extrapolations yield dissociation constants of 2-3 pM for both reduced coenzymes. The nonactive sites are occupied with dissociation constants of 57 pM for NADH and 700 pM for NADPH. These data indicate that glutamate has only a small effect on the binding of the reduced coenzyme to the nonactive site.

86 citations