DNA amplification method tolerant to sample degradation.

Gang Wang; Elizabeth Maher; Cameron Brennan; Lynda Chin; Christopher Leo; Manjit Kaur; Penny Zhu; Martha Rook; Jia Liu Wolfe; G Mike Makrigiorgos

doi:10.1101/gr.2813404

PubMed · 15520297

DNA amplification method tolerant to sample degradation.

Gang Wang·Elizabeth Maher·Cameron Brennan·Lynda Chin·Christopher Leo·Manjit Kaur·Penny Zhu·Martha Rook·Jia Liu Wolfe·G Mike Makrigiorgos

View original source ↗Export metadata ↓

Abstract

Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R(2) = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.

Explore related subjects

Cell Line, Tumor Clinical Laboratory Techniques DNA Primers DNA, Circular DNA, Neoplasm Gene Expression Profiling Genome Genotype Humans Microsatellite Repeats Molecular Probe Techniques Nucleic Acid Amplification Techniques Oligonucleotide Array Sequence Analysis Specimen Handling Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Gang Wang, Elizabeth Maher, Cameron Brennan, Lynda Chin, Christopher Leo, Manjit Kaur, Penny Zhu, Martha Rook, Jia Liu Wolfe, G Mike Makrigiorgos. 2004. DNA amplification method tolerant to sample degradation.. https://doi.org/10.1101/gr.2813404

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Dissection of mammalian replicators by a novel plasmid stability assay.

A plasmid, bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR) was previously shown to be efficiently amplified to high copy number in mammalian cells and to generate chromosomal homogeneously staining regions (HSRs). The amplification mechanism was suggested to entail a head-on collision at the MAR between the transcription machinery and the hypothetical replication fork arriving from the IR, leading to double strand breakage (DSB) that triggered HSR formation. The experiments described here show that such plasmids are stabilized if collisions involving not only promoter-driven transcription but also promoter-independent transcription are avoided, and stable plasmids appeared to persist as submicroscopic episomes. These findings suggest that the IR sequence that promotes HSR generation may correspond to the sequence that supports replication initiation (replicator). Thus, we developed a "plasmid stability assay" that sensitively detects the activity of HSR generation in a test sequence. The assay was used to dissect two replicator regions, derived from the c-myc and DHFR ori-beta loci. Consequently, minimum sequences that efficiently promoted HSR generation were identified. They included several sequence elements, most of which coincided with reported replicator elements. These data and this assay will benefit studies of replication initiation and applications that depend on plasmid amplification.

Cell Line, Tumor↗

PubMed2007

Induced and repressed genes after irradiation sensitizing by pentoxyphylline.

Aim in cancer therapy is to increase the therapeutic ratio eliminating the disease while minimizing toxicity to normal tissues. Radiation therapy is a main component in targeting cancer. Radiosensitizing agents like pentoxyphylline (PTX) have been evaluated to improve radiotherapy. Commonly, cells respond to radiation by the activation of specific early and late response genes as well as by inhibition of genes, which are expressed under normal conditions. A display of the genetic distinctions at the level of transcription is given here to characterize the molecular events underlying the radiosensitizing mechanisms. The method of suppression subtractive hybridization allows the visualization of both induced and repressed genes in irradiated cells compared with cells sensitized immediately after irradiation. The genes were isolated by cDNA-cloning, differential analysis and sequence similarity search. Genes involved in protein synthesis, metabolism, proteolysis and transcriptional regulation were detected. It is important that genes like KIAA280, which were only known as unidentified EST sequences before without function, but inaccessible by array technology were recovered as functional genes. Database searches for PTX-induced genes detected a human mRNA completely unknown. In case of suppressed genes, we detected several mRNAs; one thereof shows homology to a hypothetical protein possibly involved in signal transduction. A further mRNA encodes the protein BM036 supposed to associate with the E2F transcription factor. A hypothetical protein H41 was detected, which may repress the Her-2/neu receptor influencing breast cancer, gliomas and prostate tumors. Radiation combined with PTX may lead to a better prognosis by down regulation of the Her-2/neu, which will be proven by clinical studies in the near future.

Cell Line, Tumor↗

PubMed2007

Laser microfabrication of hydroxyapatite-osteoblast-like cell composites.

We have developed a novel approach for layer-by-layer growth of tissue-engineered materials using a direct writing process known as matrix assisted pulsed laser evaporation direct write (MAPLE DW). Unlike conventional cell-seeding methods, this technique provides the possibility for cell-material integration prior to artificial tissue fabrication. This process also provides greater flexibility in selection and processing of scaffold materials. In addition, MAPLE DW offers rapid computer-controlled deposition of mesoscopic voxels at high spatial resolutions. We have examined MAPLE DW processing of zirconia and hydroxyapatite scaffold materials that can provide a medical device with nearly inert and bioactive implant-tissue interfaces, respectively. We have also demonstrated codeposition of hydroxyapatite, MG 63 osteoblast-like cells, and extracellular matrix using MAPLE DW. We have shown that osteoblast-like cells remain viable and retain the capacity for proliferation when codeposited with bioceramic scaffold materials. Our results on MG 63-hydroxyapatite composites can be extended to develop other integrated cell-scaffold structures for medical and dental applications.

Cell Line, Tumor↗