Multiple displacement amplification for malaria parasite DNA

Multiple displacement amplification (MDA) using Phi29 has proved to be an efficient, high-fidelity method for whole genome amplification in many organisms. This project was designed to evaluate this approach for use with the malaria parasite Plasmodium falciparum. In particular, we were concerned that the AT richness and presence of contaminating human DNA could limit efficiency of MDA in this system. We amplified 60 DNA samples using phi29 and scored 14 microsatellites, 9 single-nucleotide polymorphisms (SNPs), and gene copy number at GTP-cyclohydrolase I both before and after MDA. We observed 100% concordance in 829 microsatellite genotypes and in 499 SNP genotypes. Furthermore, copy number estimates for the GTP-cyclohydrolase I gene were correlated (r(2) = 0.67) in pre- and postamplification samples. These data confirm that MDA permits scoring of a range of different types of polymorphisms in P. falciparum malaria and can be used to extend the life of valuable DNA stocks.     

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.     

SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification

Whole genome amplification by multiple displacement amplification (MDA) offers investigators using precious genomic DNA samples a high fidelity method for amplifying nanogram quantities of DNA several thousandfold. This becomes especially important for the modemrn day genomics researcher who more and more commonly is applying today's genome scanning technologies to patient cohort samples collected years ago that are irrecoverable and invariably in short supply. We present evidence here that MDA-prepared genomic DNA includes artifacts of chromosomal copy number that resemble copy number polymorphisms (CNPs) upon analysis of the DNA on the Affymetrix 10K GeneChip. The study of CNPs in both health and disease is a rapidly growing area of research, however our current understanding of the relevance of CNPs is incomplete. Our data indicate that utilization of whole genome-amplified samples for analysis heavily reliant on accurate copy number retention could be confounded if the genomic DNA sample was subjected to MDA. We recommend that small amounts of patient cohort DNA stocks be set aside and not subjected to whole genome amplification in order to facilitate the unbiased determination of chromosomal copy numbers when desired.     

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.     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

Multiple replication origins are used during Drosophila chorion gene amplification

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.     

Multiple differentiation of clonal teratocarcinoma stem cells following embryoid body formation in vitro

We have described the differentiation in vitro of clonal pluripotent teratocarcinoma stem cells derived from isolated single cells. By using solvent-resistant plastic petri dishes as a substratum for cell growth, it is possible to prepare histological sections of the cultures which can be compared with sections of teratocarcinomas formed in vivo by the same cells. Our results indicate that almost all of the cell types found in the tumors are formed in vitro, including cartilage, keratinizing epithelium, pigmented epithelium, neural tissue, and muscle. The cells are organized in a tissue structure which is remarkably similar to that found in vivo.     

Multivariate phenotype analysis enables genome-wide inference of mammalian gene function

The function of the majority of genes in the human and mouse genomes is unknown. Investigating and illuminating this dark genome is a major challenge for the biomedical sciences. The International Mouse Phenotyping Consortium (IMPC) is addressing this through the generation and broad-based phenotyping of a knockout (KO) mouse line for every protein-coding gene, producing a multidimensional data set that underlies a genome-wide annotation map from genes to phenotypes. Here, we develop a multivariate (MV) statistical approach and apply it to IMPC data comprising 148 phenotypes measured across 4,548 KO lines.There are 4,256 (1.4% of 302,997 observed data measurements) hits called by the univariate (UV) model analysing each phenotype separately, compared to 31,843 (10.5%) hits in the observed data results of the MV model, corresponding to an estimated 7.5-fold increase in power of the MV model relative to the UV model. One key property of the data set is its 55.0% rate of missingness, resulting from quality control filters and incomplete measurement of some KO lines. This raises the question of whether it is possible to infer perturbations at phenotype–gene pairs at which data are not available, i.e., to infer some in vivo effects using statistical analysis rather than experimentation. We demonstrate that, even at missing phenotypes, the MV model can detect perturbations with power comparable to the single-phenotype analysis, thereby filling in the complete gene–phenotype map with good sensitivity.A factor analysis of the MV model’s fitted covariance structure identifies 20 clusters of phenotypes, with each cluster tending to be perturbed collectively. These factors cumulatively explain 75% of the KO-induced variation in the data and facilitate biological interpretation of perturbations. We also demonstrate that the MV approach strengthens the correspondence between IMPC phenotypes and existing gene annotation databases. Analysis of a subset of KO lines measured in replicate across multiple laboratories confirms that the MV model increases power with high replicability.

The function of the majority of genes in the human and mouse genomes is unknown, and illuminating this "dark genome" is a major challenge for the biomedical sciences. This study shows that multi-dimensional phenotypes from single-gene knockout mouse lines can be analysed at a genome-wide scale both to increase power and infer missing phenotypes.     

Multiple synaptinemal complexes at the region of gene amplification in Acheta

Ovaries of Acheta domesticus (house cricket) were fixed for electron microscopy at two stages of development: (1) ovaries containing mainly oocytes at interphase and early prophase of meiosis, and (2) ovaries with oocytes mainly at pachytene and diplotene. The E.M. study was accompanied by three types of light microscopy controls consisting mainly of cytochemical tests. Every oocyte contains a DNA body which at pachytene and diplotene acquires the appearance of a puff. In the light microscope two zones can be distinguished inside the body: (1) an inner core of DNA and (2) and outer shell of RNA. In the E. M. the inner core consists of a fibrillar material and the outer shell is composed of areas of high electron opacity consisting mainly of tightly packed particles and fibrils. At these stages synaptinemal complexes are seldom seen associated with the DNA body but are present throughout the nucleus as part of the paired chromosomes. The complexes are present as single units. — In the oocytes at interphase and early prophase of meiosis, where the DNA body is active in DNA replication, the body appears in the light microscope as a large Feulgen positive sphere containing Feulgen negative areas. In the E. M. at these stages the DNA body consists of: (a) the two components found at pachytene, (b) a third electron dense component which is more homogeneous than the other two, and (c) of large assemblies of synaptinemal complexes originating from several centers. The most significant features of the axial complexes are: (1) the circular packing of the complexes, (2) their occurrence in packages of 300 to 400 units and (3) the fact that not all of the DNA body forms complexes but only a part of it.Biochemical experiments (Lima-de-Faria, Birnstiel and Jaworska, 1969) have demonstrated the amplification of ribosomal cistrons in the DNA body of Acheta. The simplest explanation is that the multiple complexes are formed either between the extra gene copies of the two homologues, or between the extra copies of each chromosome as well. There seems to be a correlation between the presence of multiple axial complex formation and gene amplification in Acheta but the exact relation between the two phenomena demands further study.Dedicated to Dr. Sally Hughes-Schrader on the occasion of the seventyfifth birthday on the twentyfifth of January 1970.     

Genotyping of Bison bonasus K-casein gene following DNA sequence amplification

DNA isolated from 30 Bison bonasus blood samples was used for amplification of a kappacasein gene fragment to detect possible polymorphism. Bovine K-caseins exist in A, B and E variants. In the European bison from Poland, we have found only BB genotypes. Sequencing of the polymerase chain reaction product revealed further polymorphisms typical for B. bonasus.     

Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production.

Retroviral vectors offer major advantages for gene transfer studies but have not been useful for producing proteins in large quantities. This deficiency has resulted in part from interference to superinfection, which limits the numbers of active proviruses in cells. Recently, we found that these vectors amplify when they are added as calcium phosphate precipitates to cocultures of cells that package retroviruses into ecotropic and amphotropic host range envelopes. Helper-free virions from either cell type can infect the other without interference, resulting in theoretically limitless back-and-forth (ping-pong) vector replication. In initial studies, however, amplifications of a vector that contained the human growth hormone gene ceased when the hormone produced was 0.3% or less of cellular protein synthesis. This limit was caused by two factors. First, recombinant shutoff viruses that are replication defective and encode envelope glycoproteins form at a low probability during any round of the vector replication cycle and these spread in cocultures, thereby establishing interference. Single cells in shutoff cocultures therefore synthesize both ecotropic and amphotropic envelope glycoproteins, and they release promiscuous (presumably hybrid) virions. The probability of forming shutoff viruses before the vector had amplified to a high multiplicity was reduced by using small cocultures. Second, cells with large numbers of proviruses are unhealthy and their proviral expression can be unstable. Stable expresser cell clones were obtained by selection. Thereby, cell lines were readily obtained that stably produce human growth hormone as 4 to 6% of the total protein synthesis. A ping-pong retroviral vector can be used for high-level protein production in vertebrate cells.     

Genetic analysis of the steroid 21-hydroxylase gene following in vitro amplification of genomic DNA.

The 5′ end of the steroid 21-hydroxylase B gene encompassing putative control regions and the first 3 exons, has been selectively amplified in vitro from a number of patients with congenital adrenal hyperplasia caused by a deficiency of this enzyme. Sequence analysis has revealed a number of isolated instances of gene conversion to the 21-hydroxylase A sequence. One mutation, a C to G transversion at the 3′ end of the second intron, thought to lead to incorrect splicing of the mRNA, was found in 11 subjects all with the classical form of the disease.     

Multiple displacement amplification of isolated DNA from human gallstones: molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis

BACKGROUND: Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. MATERIALS AND METHODS: DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. RESULTS: Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. CONCLUSIONS: We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens.     

A branching process model of gene amplification following chromosome breakage.

We have devised a mathematical model of gene amplification utilizing recent experimental observations concerning dihydrofolate reductase (DHFR) gene amplification in CHO cells. The mathematical model, based on a biological model which proposes that acentric elements are the initial intermediates in gene amplification, includes the following features: (1) initiation of amplification by chromosomal breakage to produce an acentric structure; (2) replication of acentric DNA, once per cell cycle; (3) dissociation of replicated acentric DNA; (4) unequal segregation of acentric DNA fragments to daughter cells at mitosis; (5) subsequent reintegration of acentric fragments into chromosomes. These processes are assumed to be independent for each element present in a cell at a given time. Thus, processes of unequal segregation and integration may occur in parallel, not necessarily in a unique sequence, and may be reiterated in one or multiple cell cycles. These events are described mathematically as a Galton-Watson branching process with denumerable infinity of object types. This mathematical model qualitatively and quantitatively reproduces the major elements of the dynamical behavior of DHFR genes observed experimentally. The agreement between the mathematical model and the experimental data lends credence to the biological model proposed by Windle et al. (1991), including the importance of chromosome breakage and subsequent gene deletion resulting from resection of the broken chromosome ends as initial events in gene amplification.