Molecular applications of flow cytometry

Abstract

t a rge t Pr imer B re fe rence P r imer B T he detection of genetic abemations is playing a more prominent role in our understanding of disease than ever before. It is now possible to detect gene mutations, gene translocafion, gene amplification, gene deletions, and gene rearrangements with precision. In addition, the measurement of gene expression can provide a means for determining a molecular phenotype for cells. Currently most of these procedures are performed on isolated DNA or RNA where the correlation to the cells of origin cannot be identified or microscopically where the quantitative information is less reliable. Flow cytometry has the advantage of rapid cell-by-cell integration and the ability to discriminate multiple parameters simultaneously. The possibility, for example, of determining both an immunophenotype using fluorochrome-conjugated antibodies to cellular proteins and a molecular phenotype using fluorochrome-conjugated probes seems to be within our grasp. There are several approaches under development for applying flow cytometry to molecular techniques. These include sorting distinct cell populations based on immunophenotype and applying quantitative polymerase chain reaction (PCR) procedures for detection of DNA or RNA copy numbers. Only 1,000 cells are required, and correlation between the target genetic level and protein product level can be directly compared. In situ hybridization can now be reliably performed on cells attached to microscope slides or in suspension where the cells can either be viewed microscopically or analyzed by flow cytomelry. The current problem with in situ hybridization is sensitivity due to the current detection levels by flow cytometry that require an excess of 500 copies per cell. One reason for this low Figure 1. Hybrid competitive reference standard: Two competitive reference standards are first prepared having regional sequence homology with the target gene sequence of interest and a ref~ence gene sequence on the same chromosome. An SmaI restriction site is introduced using a PCR-based site-directed mutagenesis procedure. These two standards are ligated together to produce the hybrid competitive reference standard shown. Target primers A and B and refezence primers A and B are introduced for quantitation.