An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 g DNA/g (wet weight) of soil and 400 g DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample fingerprints. Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.     

Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies

We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.     

Nucleolar DNA in oocytes of Salmo irideus (Gibbons)

The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 g. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus ( = 1.731 g cm^–3) is 1.715 g cm^–3 and corresponds to a G + C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA.Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.     

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.

     

Identification of a codominant amplified polymorphic DNA marker linked to the verticillium wilt resistance gene in tomato

Resistance to verticillium wilt, a vascular disease causing yield losses in many crops, is conferred in tomato by a single dominant allele, Ve. A population segregating for the Ve allele was generated using near-isogenic tomato lines. Analysis of the parental tomato DNA using the polymerase chain reaction and 400 random primers, each 10 deoxyribonucleotides in length, produced 1,880 amplified DNA fragments. Of the four polymorphisms observed between the resistant and susceptible parental genotypes, only one was linked to the Ve gene. No recombination was observed between this DNA marker and the Ve locus, indicating that the linkage is less than 3.5±2.7 cM. The marker detected both the susceptible and resistant alleles, producing amplified DNA fragments of approximately 1,300 and 1,350 bp, respectively. The sequence of the primer, determined from cloned amplified products, was 5 CTCACATGCA 3 instead of the expected 5 CTCACATGCC 3. The marker will be of value to tomato breeding programs because of the tight linkage, Codominant nature, and analytical procedure utilized.     

Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification

Summary Deletions are the most common molecular defect in steroid sulfatase (STS) deficiency. We describe the application of multiplex DNA amplification, by polymerase chain reaction, for deletion screening in patients with STS deficiency (STS-PCR). Genomic DNA from 38 unrelated patients was amplified using two sets of primers, corresponding to the 5 and the 3 ends of the STS gene. The analysis of the amplified products was always consistent with the results obtained by Southern analysis. This method represents a sensitive fast non-radioactive test for detecting STS gene deletions.On leave from Department of Pediatrics, University of Reggio Calabria, Italy     

DNA nanotechnology with one-dimensional self-assembled nanostructures

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Highlights► Hybridization chain reactions for amplified DNA detection and DNA assembly. ► Rolling circle amplification-synthesized DNAzyme chains for amplified DNA detection. ► Bi-enzyme cascades and biocatalytic synthesis of metallic nanowires on DNA templates. ► Self-assembled one-dimensional DNA nanostructures for biocatalytic cascades. ► Programmed assembly of metal nanoparticles on one-dimensional DNA nanostructures.     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

A molecular marker associated with the H21 Hessian fly resistance gene in wheat

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (Coker 797 * 4 / Hamlet) and differed by the presence or absence of H21 (on 2RL) derived from Chaupon rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.     

Effects of sample treatments on genome recovery via single-cell genomics

Single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we show that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.Relatively little is known about the functioning of complex microbial communities, largely due to the difficulty in culturing most microbes (Rappe and Giovannoni, 2003). Although metagenomics can provide information on the genetic capabilities of the entire community, it is difficult to connect predicted gene functions to specific organisms using metagenomics (Morales and Holben, 2011). One method to address these challenges is single-cell genomics, where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA; Lasken, 2012; Blainey, 2013).When working with environmental or clinical samples it is generally impractical to work with fresh samples. Most samples need to be preserved in some way before they are studied by single-cell genomics. A possible difficulty is that preservation methods could induce damage to the DNA that would negatively impact genome recovery. The effect of treatments on the cells is especially important in single-cell genomics because it is already established that the process produces incomplete genomes even with fresh cells (Marcy et al., 2007). The average estimated genome completeness of 650 single-cell genomes that are publicly available in IMG (https://img.jgi.doe.gov) is 41%, as based on the presence of conserved single copy genes by the method in Rinke et al. (2013).The purpose of this study was to test the effects of common sample treatments on the recovery of genomes from single cells. Three methods of sample preservation were tested: cryopreservation with 20% glycerol as a cryoprotectant, preservation in 70% ethanol, and preservation in 4% paraformaldehyde. Because paraformaldehyde treatment causes crosslinks between nucleic acids and proteins, we tested cells exposed to 4% paraformaldehyde with and without having their crosslinks reversed by heat treatment. Additionally, there is demand for obtaining single-cell genomes from particular taxa within a microbial community. A common approach for this type of isolation makes use of fluorescence in situ hybridization (FISH) to identify the target cells (Podar et al., 2007; Haroon et al., 2013). Thus, cells that had undergone a typical FISH protocol without prior fixation (Yilmaz et al., 2010) were included as an additional treatment to separate the FISH protocol from the fixation steps.In order to determine if the GC content of a genome would affect the results, three bacterial strains were chosen with differing genomic G+C contents: Pedobacter heparinus DSM 2366, 42% GC; Escherichia coli K12-MG1655, 51% GC; and Meiothermus ruber DSM 1279, 63% GC. These have complete genome sequences available and have the same cell structure (Gram negative) to control for major differences in lysis efficiency. Forty cells of each of the three strains underwent each of the five treatments outlined above (Figure 1).Open in a separate windowFigure 1Schematic of experimental workflow. For clarity the treatments are color coded throughout the figure and are: blue, Cryo—cryopreservation; green, FISH—FISH treatment; yellow, EtOH—ethanol fixation; orange, PFA xlinks—paraformaldehyde fixation with crosslinks reversed; red, PFA—paraformaldehyde fixation with crosslinks intact; brown, Pos—positive controls (wells each with 100 cryopreserved cells); purple, Neg—no cell-negative controls. The layout used for the single cell isolations in a 384-well plate is shown. Each strain/treatment combination was sorted separately onto one plate per strain. The MDA step in this figure plots the Cp (inflection point) values for the real-time MDA amplification curves for all three bacterial species combined. The MDA reaction is run for 16 h, so any wells that show no amplification by that time are indicated as 16+ on the graph. Each column in the chart summarizes data from 120 separate MDA reactions, except for the negative control, which involves 60 reactions. 16S rRNA gene screening indicates the percentage of the wells for each treatment that produced a 16S rRNA gene sequence from the reference organism.All cells underwent an alkaline lysis and MDA (Woyke et al., 2011). The time at which the inflection point of the real-time amplification curves occurs (crossing point; Cp value) correlates with the completeness of the recovered single-cell genomes (Supplementary Figure S1). Cp order was ranked as follows: cryopreservation<FISH<ethanol<paraformaldehyde with crosslinks reversed<paraformaldehyde with crosslinks intact=negative controls (Figure 1). Although the real-time kinetics showed some amplification, in no cases did the MDA products from paraformaldehyde with or without crosslink reversal treatments produce a 16S rRNA gene amplicon from PCR (Figure 1) indicating that the MDA amplicons were likely due to nonspecific amplification of primers. For each strain, eight cells from each of the three treatments that produced the expected 16S rRNA gene sequence and had the earliest Cp times in the treatment (cryopreservation, ethanol, and FISH) were selected for shotgun sequencing.To eliminate any biases due to varying sequencing depth, the sequences were randomly subsampled to a coverage depth of 315 × for each single amplified genome (SAG; Supplementary Figure S2). The reads were mapped to the reference genomes, a de novo assembly was performed and the assembled data were also mapped to the reference genomes.Cryopreservation resulted in SAGs with the highest percentage of the genome recovered for all three strains of bacteria (Figure 2; Supplementary Figure S3). FISH treatment produced reduced genome coverage, and ethanol preservation resulted in the lowest amount of the genome recovered. These treatments are significantly different from each other (Figure 2; Supplementary Figure S3). Despite the trend for cells with a higher GC content to have higher MDA Cp values, there is no clear effect of GC content on the amount of genome recovered by the various treatments (Supplementary Figures S1 and S3).Open in a separate windowFigure 2(a) Coverage plots for each organism/treatment combination. Cryo—cryopreservation, EtOH—ethanol preservation, FISH—FISH treatment. The horizontal black lines in the center of each plot represent the complete reference genomes and the vertical colored lines indicate which parts of the genome were recovered in the assemblies. Redder colors indicate that more single cells contained that region in their assembly. (b) Percent of the genomes recovered for each treatment with the single cells from all three organisms averaged together and the error bars indicating one standard deviation. Light gray bars indicate the genome recovery by mapping the reads to the reference genomes. A base pair was considered to be recovered if at least 10 reads were mapped to cover it. Dark gray bars are the genome recovery by mapping contigs produced by de novo assembly to the reference genomes. The treatments are significantly different from each other (ANOVA; P<0.0001).The lack of amplicons from cells treated with paraformaldehyde is likely due to the crosslinks preventing the phi29 polymerase from accessing the DNA. The heat treatment to reverse the crosslinks was either insufficient to reverse them or resulted in DNA strand breakage and depurination that damaged the DNA sufficiently that it was unable to be amplified.The FISH process is intended to improve access of the oligo probe to its RNA or DNA target site in the cells. As this might result in greater access of the phi29 polymerase to the DNA, one would expect the MDA from this treatment to be the most efficient. However, the fact that the MDA kinetics were delayed and that the genome recovery was reduced compared to the cryopreserved samples indicates that other factors must be involved. As the washing steps should remove the components of the FISH buffers prior to MDA, they would not directly affect the MDA process. Thus, the reduced genome recovery is likely derived from damage to the DNA during the FISH treatment. Since the FISH process can denature high-AT regions of DNA at the temperatures used, the single-stranded DNA that results could be more susceptible to damage (Blake and Delcourt, 1996). This interpretation is supported by the genome recovery in the low-GC-organism P. heparinus being lower than in the other two organisms (Supplementary Figure S3). In addition, it has been shown that formamide can degrade purine nucleosides (Saladino et al., 1996). Small amounts of damage could explain the reduction in the genome recovered by the MDA.The significant reduction in genome recovery from ethanol-preserved cells is challenging to explain. It is unlikely that ethanol could carry over and inhibit the MDA reaction because the cells were washed twice before sorting and the sorting process itself results in significant dilution of the sample (Rodrigue et al., 2009). Since there is no known mechanism for ethanol to damage the DNA, the substantial reduction in the amount of the genome recovered must be due to the polymerase having restricted access to the DNA. Ethanol causes proteins to denature and precipitate (Yoshikawa et al., 2012). These could aggregate around the DNA and prevent access by the polymerase.Although single-cell genomics has great potential to provide insight into the vast number of microbes that have not been cultivated, sample handling can greatly impact the completeness of single-cell genomes. Our results suggest that samples that have been archived by preservation in paraformaldehyde will be unsuitable for the production of single-cell genomes and that ethanol-preserved samples are likely to produce single-cell genomes of reduced quality. Thus, we recommend use of cryopreserved specimens for best results and fixation-free FISH (Yilmaz et al., 2010; Haroon et al., 2013) if targeted flow sorting is to be employed.     

Detection of β-thalassemia mutations in the Chinese using amplified DNA from dried blood specimens

Summary This paper describes DNA polymerase chain reaction (PCR) amplification directly from dried blood specimens for the detection of the -thalassemia mutation in China. Target DNA was amplified to span the -globin gene regions, which included ten types of mutation sites specific for Chinese -thalassemias. Ten kinds of oligonucleotide probes were constructed and used to hybridize with the amplified DNA. A total of 170 -thalassemia alleles originating from eastern, southwestern and southern China were analyzed. The results revealed that the distributions of different types of mutations were different in the three regions. The most common types in southern China were a frameshift at codons 41/42 and a CT substitution at IVS II n.654, the most frequent types in south-western China were codon 17 and IVS II n.654 mutations, and the predominant mutations in eastern China were frameshifts at codons 41/42 and 71/72.     

Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using φ29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, “Magnetospirillum magneticum AMB-1,” whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.Magnetotactic bacteria synthesize nanosized intracellular magnetic particles, also referred to as magnetosomes, by accumulating iron ions from the environment. Since the first report on the identification of magnetotactic bacteria (2), the morphological and phylogenetic diversity of these organisms has been observed in various aquatic environments (12, 25, 27, 30). However, bacterial strains available in pure culture are currently limited to a few genera. Desulfovibrio magneticus strain RS-1 is the only isolate of magnetotactic bacteria that is classified among the Deltaproteobacteria (13, 23), while Magnetospirillum spp., marine magnetic vibrio strain MV-1, and “Magnetococcus strain MC-1” are phylogenetically affiliated within the Alphaproteobacteria group (24, 27). This limitation is mainly because not much is known about their metabolic requirements, culturing conditions, and obligate coculture requirements.Isolation and enrichment of magnetotactic bacteria are generally conducted by applying a magnetic field to a container containing a sediment sample from the environment. The capillary racetrack method is a highly selective enrichment technique that separates magnetotactic bacteria from other contaminants (31). The magnetic separation method that involves the use of a large glass apparatus is efficient and suitable for analyzing samples containing more than 100 ml of sediment and water (12, 16). These techniques have been applied to investigate community structure and phylogenetic diversity of uncultured magnetotactic bacteria in the environment based on 16S rRNA analyses (3, 7, 26, 29). In a recent study, DNA isolation enabling gene cloning was examined by magnetically collecting a large number of magnetotactic cells from environmental samples, and two gene fragments, probably containing parts of magnetosome islands (MAIs) derived from magnetotactic bacteria of the Alphaproteobacteria, were identified (12). However, this approach allows only for sequence gene information to be obtained from a heterogeneous bacterial community in the sample.Multiple displacement amplification (MDA) can generate microgram quantities of high-quality DNA sample from a few femtograms of DNA template (5, 6). We previously revealed that MDA is a powerful tool for whole-genome amplification from the metagenome of an uncultured bacterial community (32). Studies have been conducted to determine the efficacy of MDA for analyzing genomic DNA preparations from a limited number of bacterial cells (14, 17, 21, 22, 28). Complete genomic sequencing of an uncultured gut symbiont in termites has been achieved using MDA products amplified from approximately 1,000 cells (9). Partial genome sequencing using MDA products from a single uncultured cell has also been reported (17, 22). Such targeted genome analyses using MDA products from a single cell or genetically identical microorganisms is advantageous because it allows the assignment of individual genes to the corresponding microorganisms.In this study, an improved genome preparation method involving racetrack purification and flow cytometry followed by MDA was investigated by using a small number of uncultured magnetotactic bacteria. This method can be used for the identification of new genes from rare magnetotactic bacteria in environmental samples.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

Background  

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them.     

DNA Yields and Optimization of RAPD Patterns Using Spruce Embryogenic Lines,Seedlings, and Needles

Genetic analysis of plants relies on high yields of pure DNA samples. In conifers, such DNA samples are usually derived from tissue cultures. However, it is labourious to use embryogenic cultures for projects that require large sample numbers because the starting material has to be initiated from the seed in vitro. As an alternative, we have used seedlings as source material for DNA extraction and have obtained yields and purities comparable to embryogenic cultures. RAPD profiles obtained with DNA from embryogenic cultures and seedlings were similar and highly reproducible. Efficient and consistent amplification was achieved using primer concentrations ranging from 1.6 and 6.4 M with 0.1 to 1.0 g of template DNA. The intensity and resolution of faint bands resulting from non-optimized conditions were easily improved through further rounds of synthesis in a fresh PCR with amplified DNA diluted 500-fold.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5 terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3 terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.     

Detection of free cytoplasmic circles of transposon Tn9 multimers in Escherichia coli

Extrachromosomal circular DNA molecules consisting of IS1-cat repeats, (IS1-cat)_n, were isolated from an E. coli strain harboring nearly 30 copies of tandemly amplified transposon Tn9 located on the chromosome. The DNA circles were characterized by restriction analysis followed by Southern blotting and electron microscopic examination. Their size varied from approximately 5.5 kb to 53 kb.