2D-PCR: a method of mapping DNA in tissue sections

Abstract

A novel approach was developed for mapping the location of target DNA in tissue sections. The method combines a high-density, multi-well plate with an innovative single-tube procedure to directly extract, amplify, and detect the DNA in parallel while maintaining the two-dimensional (2D) architecture of the tissue. A 2D map of the gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was created from a tissue section and shown to correlate with the spatial area of the sample. It is anticipated that this approach may be easily adapted to assess the status of multiple genes within tissue sections, yielding a molecular map that directly correlates with the histology of the sample. This will provide investigators with a new tool to interrogate the molecular heterogeneity of tissue specimens.

Fig. 1

2D-PCR is achieved by transferring a tissue section vertically into a multi-well array device, isolating tissue subregions. The DNA is extracted and then amplified by the polymerase chain reaction. A detection map is created by imaging the amplified DNA, for example using a DNA-intercalating dye.

Fig. 2

Experimental protocol used to demonstrate the 2D-PCR concept. Tissue is transferred into wells, isolated, and lysed to extract the genomic DNA (represented by “x”s). PCR is performed to amplify the target DNA, and the targets are visualized with fluorescent dye (indicated by vertical lines “|”).

Fig. 3

Image of a breast tissue section (stained for visualization with Eosin Y) after transfer onto the multi-well device, and the visualization map created by fluorescent DNA detection with SYBR Green I after amplification by 2D-PCR. The tissue position is indicated by the outline.

Fig. 4

(A) The wells chosen for electrophoretic validation of the fluorescent signal. (B) In wells containing tissue (lanes 1–6) there was a bright fluorescent signal corresponding to detection of a 167 bp GAPDH genomic product, while negative wells (lanes 7–12) had no amplification of the 167 bp target and only weak bands of ~50 bp primer dimers, an artifact from PCR.

Fig. 5

Silicon substrate having a 1 cm² area with 100 μm diameter holes spaced 100 μm apart. The surface was pretreated with BSA and spotted with water containing food dye for visualization (dark area in center). The wells filled completely by capillary action.