An efficient resampling method for assessing genome-wide statistical significance in mapping quantitative trait Loci

Assessing genome-wide statistical significance is an important and difficult problem in multipoint linkage analysis. Due to multiple tests on the same genome, the usual pointwise significance level based on the chi-square approximation is inappropriate. Permutation is widely used to determine genome-wide significance. Theoretical approximations are available for simple experimental crosses. In this article, we propose a resampling procedure to assess the significance of genome-wide QTL mapping for experimental crosses. The proposed method is computationally much less intensive than the permutation procedure (in the order of 10(2) or higher) and is applicable to complex breeding designs and sophisticated genetic models that cannot be handled by the permutation and theoretical methods. The usefulness of the proposed method is demonstrated through simulation studies and an application to a Drosophila backcross.     

An AFLP-based genome-wide mapping strategy

To efficiently determine the chromosomal location of phenotypic mutants, we designed a genome-wide mapping strategy that can be used in any crop for which a dense AFLP (Amplified Fragment Length Polymorphism) map is available or can be made. The AFLP technique is particularly suitable to initiate map-based cloning projects because it detects many markers per reaction. First a standard set of AFLP primer combinations that results in a framework of AFLP markers well dispersed over the genome is selected. These primer combinations are applied to a limited number of mutant individuals from a segregating population to register linkage and non-linkage of the AFLP markers to the gene-of-interest. Further delineation of the area of interest is accomplished by analyzing the remaining recombinants and additional mutant individuals with AFLP markers that lie within the identified region. We illustrate the usefulness of the method by mapping three rotunda (ron) leaf-form mutant loci of Arabidopsis thaliana and show that in the initial phase of map-based cloning projects a 400–600 kb interval can be identified for the average mutant locus within a few weeks. Once such an area is identified and before initiating the more time-consuming fine-mapping procedure, it is essential to examine publicly available databases for candidate genes and known mutants in the identified region. The 390-kb interval on chromosome 4 that harbors the ron2 mutation, also carries a known flower mutant, leunig (lug); upon crossing, the two mutants appeared to be allelic. When no such candidates are found, the mapping procedure should be continued. We present a strategy to efficiently select recombinants that can be used for fine mapping.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by R. Hagemann     

An efficient and scalable extraction and quantification method for algal derived biofuel

Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids from live cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GC–FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GC–MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived from the residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae; the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris. The method is shown to be robust, highly reproducible, and fast, allowing for multiple samples to be analyzed throughout the time course of culturing, thus providing time-resolved information regarding lipid quantity and quality. Total time from harvesting to obtaining analytical results is less than 2 h.     

An efficient method for total RNA extraction from peanut seeds

Most of conventional RNA extraction methods failed to extract highly pure and integral RNA from peanut seeds because peanut seeds are extremely rich in lipids, proteins, polysaccharides, and phenolic compounds. Here, we describe a new method, named Peanut Improved Modified RNA extraction method (PIMRNAext), using SDS, Tris-saturated phenol, NaCl, and sarkosyl during the extraction process, which are particularly successful for total RNA extraction from lipid- and polysaccharide-rich materials. The proposed PIMRNAext method is simple and fast. It requires only conventional reagents and is completed within 2 h. Using PIMRNAext gives very good yields of high quality peanut RNA. This method is about ten times more efficient than conventional methods, and the RNA produced by it is compatible with further molecular biology experiments, such as RT-PCR. We propose to use the PIMRNAext method to extract RNA from peanuts and peanut-like plant species not only for RT-PCR, but also for most molecular biology techniques that need copies of pure RNA, such as microarray or cDNA library construction.     

An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs

RNA ligation has been a powerful tool for incorporation of cross-linkers and nonnatural nucleotides into internal positions of RNA molecules. The most widely used method for template-directed RNA ligation uses DNA ligase and a DNA splint. While this method has been used successfully for many years, it suffers from a number of drawbacks, principally, slow and inefficient product formation and slow product release, resulting in a requirement for large quantities of enzyme. We describe an alternative technique catalyzed by T4 RNA ligase instead of DNA ligase. Using a splint design that allows the ligation junction to mimic the natural substrate of RNA ligase, we demonstrate several ligation reactions that appear to go nearly to completion. Furthermore, the reactions generally go to completion within 30 min. We present data evaluating the relative importance of various parameters in this reaction. Finally, we show the utility of this method by generating a 128-nucleotide pre-mRNA from three synthetic oligoribonucleotides. The ability to ligate synthetic or in vitro transcribed RNA with high efficiency has the potential to open up areas of RNA biology to new functional and biophysical investigation. In particular, we anticipate that site-specific incorporation of fluorescent dyes into large RNA molecules will yield a wealth of new information on RNA structure and function.     

A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization

To rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5′-Cm⁵CGG-3′ sites in the genome, visualized by fluorescence polarization (FP) based measurement of DNA methylation (FPDM). In FPDM, the methyl-sensitive HpaII and methyl-insensitive MspI restriction enzymes were employed to achieve DNA cleavage, followed by incorporation of fluorescent dCMP into the enzyme-cleavage products through polymerase chain extension, yielding an FP-ratio between the HpaII- and MspI-restricted preparations as a measure of methylation. FPDM provided stable estimates of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII/MspI restriction site in accord with, and more accurate than, determination by gel electrophoresis. FPDM was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC.     

Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site.

Processing at the L3 polyadenylation site of human adenovirus-2 involves endonucleolytic cleavage generating the 3' terminal sequence -UAOH to which adenosine residues are added. This dinucleotide is 19 nucleotides downstream of the AAUAAA polyadenylation signal. The ATP analog cordycepin triphosphate (3' dATP) inhibits poly(A) synthesis, but precursor RNA is processed to give a product terminating in -UAAH. Addition of only one adenosine analog demonstrates that the initial poly(A) tract is synthesized by polymerization of single residues rather than by ligation of preformed poly(A). Cleavage is not coupled to polyadenylation since incubation with an ATP analog containing a non-hydrolyzable alpha--beta bond generates a product with a 3' terminus coincident with the -UAOH) addition site. Addition of this accurately processed RNA to a nuclear extract results in efficient polyadenylation, suggesting that downstream sequences are not required for synthesis of the poly(A) tract. Finally, processing at the L3 poly(A) site may involve both endonucleolytic and exonucleolytic activities.     

A simple method for DNA restriction site mapping.

When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus. ta restriction map can then be constructed from an analysis of the size distribution of these molecules. This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.     

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.     

An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.     

Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viral RNA template during RNA replication

The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5' ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.