Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Methylation profiling using degenerated oligonucleotide primer-PCR specific for genome-wide amplification of bisulfite-modified DNA

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample, whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles, we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP, CTCGAGCTGHHHHHAACTAC, where H is a mixture of base consisting of 50% A, 25% T, and 25% C; and second 5' DOP, CTCGAGCTGDDDDDGTTTAG, where D is a mixture of base consisting of 50% T, 25% G, and 25% A. Our results showed that bisulfite-modified DNAs from a cell line, cord blood cells, or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template.     

The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

A Whole Genome Amplification Method to Generate Long Fragments from Low Quantities of Genomic DNA

Several whole genome amplification strategies have been developed to preamplify the entire genome from minimal amounts of DNA for subsequent molecular genetic analysis. However, none of these techniques has proven to amplify long products from very low (nanogram or picogram) quantities of genomic DNA. Here we report a new whole genome amplification protocol using a degenerate primer (DOP-PCR) that generates products up to about 10 kb in length from less than 1 ng genomic template DNA. This new protocol (LL-DOP-PCR) allows in the subsequent PCR the specific amplification, with high fidelity, of DNA fragments that are more than 1 kb in length. LL-DOP-PCR provides significantly better coverage for microsatellites and unique sequences in comparison to a conventional DOP-PCR method.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

染色体显微切割与DOP—PCR结合对赤麂Sry基因克隆，测序及初步定位

应用显微切割技术获得赤麂1号,Y1,Y2染色体,通过DOP－PCR增加模板DNA拷贝数,然后用人的性别决定基因（Sex-tetermininig Region of the Chromosome Y,SRY)中HMG框内设计1对引物,对DOP－PCR产物进行扩增,在雄性赤麂Y2染色体DOP－PCR产物中扩增出与人SRY基因同源的Sry基因片段,克隆,测序,首次在分子水平上证明赤麂Y2染色体是真正的Y染色体,同时对赤麂Syr基因进行了初步定位。     

杉木第四号染色体特异性RAPD片段的获得

分别分离杉木(Cunninghamia lanceolata(Lamb.)Hook)同一细胞中的第四号具随体染色体及剩余20条非随体染色体,进行DOP-PCR扩增,分别以随体染色体及非随体染色体的DOP-PCR产物为模板,用成对随机引物进行RAPD分析。用引物对OPB07+OPB10进行扩增,在500-250 bp之间,随体染色体有4条特异扩增带;用引物对OPB07+OPB18在900 bp左右获得1条随体染色体特异带;用引物对OPD07+OPD05在250 bp左右得到随体染色体1条特异扩增带。     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”