Degenerate Oligonucleotide primed-PCR Products Research Articles

Genome-wide scan of the soybean genome using degenerate oligonucleotide primed PCR: an example for studying large complex genome structure

The availability of next generation sequencing instruments has made large-scale and genome-wide sequence analysis more feasible in a wild variety of species with large complex genomes, especially crops. This report suggested an approach for characterizing large complex genomes of less-studied/orphan crops. Degenerate oligonucleotide primed PCR (DOP-PCR) is a useful tool for the survey of genomes in less-studied/orphan crops, as prior sequence information is not necessary. Here, four different degenerate primers were redesigned from previously described DOP-PCR primers. The degeneracy of these primers was increased with the addition of two more ‘Ns’. The amplified DOP-PCR products from Sinpaldalkong 2, a soybean genotype, were applied to GS-FLX and the reads from Sinpaldalkong 2 were mapped against Williams 82 as a reference (http://www.phytozome.net/soybean.php), using the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/). These results suggest the identification of 4 single nucleotide polymorphisms between Sinpaldalkong 2 and Williams 82 and recent duplication of the soybean genome. The sequenced reads were subsequently assembled into contigs by Newbler under default conditions. A total of 29 Sinpaldalkong 2 contigs exhibited 95% similarity and < E-100 when mega-blasted with Williams 82 reference sequences. These contigs were mapped to the soybean chromosomes and positioned as clusters within each chromosome. Most of the contigs also showed similarity with the Arabidopsis RNase H domain-containing protein, suggesting a potential way to study retrotransposons in less-studied/orphan crops. Using these modified DOP-PCR primers and GS-FLX, it is possible to obtain insight into the large complex genomes of less-studied/orphan crops.

Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes

BackgroundChromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level.ResultsWe report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei.ConclusionsAtomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.

DcDegenerate Oligonucleotide Primed-PCR for Multilocus, Genome-wide Analysis From Limited Quantities of DNA

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples (<or= 0.100 ng). Experiments involved altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases. Increasing the degeneracy of the primer and the number of cycles that use a low annealing temperature should improve the nonspecific amplification of the DOP-PCR reaction. The addition of proofreading enzymes should allow for longer amplification products, increasing the genome coverage of the reaction. Low-input DNA quantities were examined for the primer and the cycle number studies using standard DOP-PCR parameters. The optimized DOP-PCR technique was then implemented for the polymerase study. All DOP-PCR products were amplified by using a multiplex microsatellite amplification kit to evaluate products from multiple chromosomes, followed by separation and detection by capillary electrophoresis. The 10 N primer, 12 nonspecific cycles, and the addition of the DeepVent proofreading enzyme all significantly increased the number of short tandem repeat alleles successfully amplified. All modifications also lowered the rate of allele drop-in, or sporadic additional allele occurrence, when compared with DOP-PCR results published earlier. Further, an average of > 0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results show that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples.

Individual identification by DNA polymorphism using formalin‐fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin-fixed material is very limited. In the present study the use of 6 week formalin-fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin-fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin-fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide-primed PCR (DOP-PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP-PCR products. Furthermore, large molecular size fragments were purified from the DOP-PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.

Use of degenerate oligonucleotide primed PCR (DOP-PCR) for the genotyping of low-concentration DNA samples.

Degenerate oligonucleotide primed amplification (DOP-PCR) is an efficient method for performing whole genome amplification. We analysed the yield of DNA using this technique starting with various quantities of material. We used DOP-PCR products to genotype single nucleotide polymorphisms and insertion/deletion polymorphisms. DOP-PCR also proved usable for SSCP analysis.

Isolation of chromosome-specific paints from high-resolution flow karyotypes of the sheep (Ovis aries).

High-resolution bivariate flow karyotypes were obtained using fibroblast cell lines from a sheep with a normal karyotype (2n = 54), from sheep carrying Robertsonian translocation chromosomes and from sheep-hamster somatic cell hybrids. By taking advantage of the presence of chromosome polymorphisms, translocation chromosomes and sheep-hamster somatic cell hybrids, all sheep chromosomes were isolated by flow sorting. Chromosome-specific paints were generated from each sorted peak using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The sheep chromosome present in each peak was identified by chromosome-specific microsatellite analysis of the DOP-PCR products and fluorescence in situ hybridization (FISH) onto DAPI-banded sheep metaphase chromosomes. The chromosome-specific DNA obtained in this study can be used for the production of genomic libraries and as a resource for mapping randomly cloned DNA sequences that will greatly aid the construction of genetic and physical maps in the sheep. The chromosome-specific paints will facilitate chromosome identification and contribute to the study of karyotype evolution in the sheep and related species.

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.