|Matthews, Benjamin - Ben|
Submitted to: Biotechniques
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/17/2000
Publication Date: N/A
Citation: Interpretive Summary: All genetic manipulations of plants first begin with the extraction and isolation of purified DNA, which contains the genetic blueprint of the living organism. DNA isolation in the past has usually involved a multistep process that uses hazardous chemicals and takes several days to complete. We are applying a new technology to plants in which the plant leaf is simple crushed onto a specially treated paper and a small amount of plant juice containing the DNA is preserved on the paper. A simple paper punch from this paper stained with plant juice can be analyzed by a procedure known as a polymerase chain reaction (PCR) which is a common technique used in plant molecular research. Thus, in a matter of minutes, we can isolate and prepare plant samples for DNA analysis using the new paper FTA technology. Because the procedure is so simple and the DNA paper samples can be stored at room temperature, this technique has widespread applications for detecting and analyzing DNA in crop plants.
Technical Abstract: We have developed and applied a simple procedure for the isolation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper and the genomic DNA was purified using simple, non-organic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome was detected by PCR amplification of DNA extracted from the FTA paper. DNA amplification was successful using extracts from seventeen dicot and monocot plants. Detailed studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. By increasing the number of cycles of DNA amplification, we were able to detect the GUS gene in transgenic tobacco and rice leaves using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plan genes from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.