A Short and Simple Improved-Primer Extension Preamplification (I-PEP) Procedure for Whole Genome Amplification (WGA) of Bovine Cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS / GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (～3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.     

A simple and economical method for improving whole genome alignment

Background

The recent advancement of whole genome alignment software has made it possible to align two genomes very efficiently and with only a small sacrifice in sensitivity. Yet it becomes very slow if the extra sensitivity is needed. This paper proposes a simple but effective method to improve the sensitivity of existing whole-genome alignment software without paying much extra running time.

Results and conclusions

We have applied our method to a popular whole genome alignment tool LAST, and we called the resulting tool LASTM. Experimental results showed that LASTM could find more high quality alignments with a little extra running time. For example, when comparing human and mouse genomes, to produce the similar number of alignments with similar average length and similarity, LASTM was about three times faster than LAST. We conclude that our method can be used to improve the sensitivity, and the extra time it takes is small, and thus it is worthwhile to be implemented in existing tools.

     

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Metagenomics approaches have been developing rapidly in marine sciences. However, the application of these approaches to marine eukaryotes, and in particular to the smallest ones, is challenging because marine microbial communities are dominated by prokaryotes. One way to circumvent this problem is to separate eukaryotic cells using techniques such as single-cell pipetting or flow cytometry sorting. However, the number of cells that can be recovered by such techniques remains low and genetic material needs to be amplified before metagenomic sequencing can be undertaken. In this methodological study, we tested the application of whole-genome amplification (WGA) to photosynthetic eukaryotes. We performed various optimization steps both on a mixture of known microalgal strains and on natural photosynthetic eukaryote populations sorted by flow cytometry. rRNA genes were used as markers for assessing the efficiency of different protocols. Our data indicate that WGA is suitable for the amplification of photosynthetic eukaryote genomes, but that biases are induced, reducing the diversity of the initial population. Nonetheless, this approach appears to be suitable for obtaining metagenomics data on microbial eukaryotic communities.     

A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS)

A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.     

Decontamination of MDA reagents for single cell whole genome amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.     

Genotypic analysis of multiple loci in somatic cells by whole genome amplification.

To screen multiple loci in small purified samples of diploid and aneuploid cells a PCR-based technique of whole genome amplification was adapted to the study of somatic lesions. DNA samples from different numbers of flow-sorted diploid and aneuploid cells from biopsies were amplified with a degenerate 15mer primer. Aliquots of these reactions were then used in locus-specific reactions using a single round of PCR cycles with individual sets of primers representing polymorphic markers for different regions. As a result, polymorphic markers for different chromosomal regions, including VNTRs and dinucleotide repeats, can be used to perform up to 30 locus-specific PCR assays with a single sample obtained from fewer than 1000 cells.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative amplification of its genome by multiple-displacement amplification

Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 10(7) cells). We obtained sufficient amounts (8 to 10 microg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.     

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6–3.0 Mb in single cells.     

An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening

We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.     

Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation.

Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes.     

A simple and rapid procedure for sequencing long (40-kb) DNA fragments

A simple procedure has been developed for sequencing long (40-kb) DNA fragments by the dideoxy method. The fragment is cloned in the sequencing cosmid pAA113X by in vitro packaging, and subdivided by a series of overlapping IS1-promoted deletions. The deletions are isolated by positive selection for galactose resistance. Plasmids from several thousand galactose-resistant colonies are fractionated on an agarose gel, and DNA from each fraction is restricted with enzymes (such as SphI and SalI) to shorten each deletion from the opposite end. As a result, a series of short overlapping segments, spread across the entire length of the fragment, are fused to IS1. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an IS1 primer. Sequences of IS1-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence.     

A simple and generally applicable procedure for releasing DNA from bacterial cells

Treatment of Staphylococcus simulans biovar staphylolyticus cells with acetone before digestion with lysozyme made the cells susceptible to lysis by sodium dodecyl sulfate. This technique was found to be useful for releasing DNA from a wide variety of gram-positive and gram-negative organisms.     

Designing a simple multiplex ligation-dependent probe amplification （MLPA） assay for rapid detection of copy number variants in the genome

Copy number variants （CNVs） are pervasive in the human genome and are responsible for many Mendelian diseases and genomic disorders. The detection of CNVs is an essential element of a complete mutation screening strategy. Many techniques have been developed for gene dosage testing. Multiplex ligation-dependent probe amplification （MLPA） is a robust, easy and flexible technique that can detect both deletions and duplications for more than 40 loci in one assay. It has been widely used in research and diagnostic laboratories. We routinely develop our own MLPA assays for quick validation of array comparative genomic hybridization （CGH） findings. Here we discuss the general principles and critical aspects of MLPA assay development and validation using all synthetic MLPA probes. We believe that MLPA will play important roles in the rapid detection of genomic disorders associated with genomic imbalances, the confirmation of pathogenic mutations involving exonic deletions/duplications, CNV genotyping and population frequency analysis of CNVs.     

A simple procedure for separating the subunits of ovine and bovine pituitary interstitial cell stimulating hormone

A simple, effective procedure for separating ovine or bovine interstitial cell stimulating hormone into their subunits has been described with a yield of 70–80%. The β subunit isolated by the new procedure behaves differently from that obtained by the countercurrent distribution method, and is in a more native state. In addition, full biological activity is consistently restored upon recombination of the subunits isolated by the new procedure.