A major problem in trying to understand complex developmental processes is one of heterogeneity at both the cellular and molecular levels. At the cellular level it is often difficult to identify cells that are undergoing developmental changes and to establish the stage of differentiation they have reached. At the molecular level, there is then a problem in establishing which of the many thousands of expressed genes are playing a role in development. Several approaches to identifying expressed candidate developmental genes are based on comparing the mRNA expression patterns in cells before and after transition points. Differential screening of cDNA libraries with labeled total cDNA probes from contrasting cell samples (1) provides a simple means of identifying genes that are expressed at high levels in one of the samples. cDNA subtraction protocols (2,3) have increased the sensitivity of this type of approach by removing sequences expressed in both samples. The limitation of these approaches lies in the need for large amounts of starting material necessary for cDNA preparation and subtraction. With the advent of the polymerase chain reaction (PCR) (4) and the development of techniques that allow amplification of target sequences expressed in single cells (5), the limitations of applying cDNA subtraction and differential screening have been removed.
Here we describe the details of a cDNA subtraction approach previously applied to single cells (6), which is based on the PolyAPCR method used to produce amplified cDNA representing all mRNAs present in samples as small as a single cell (7,8). PolyAPCR has been applied successfully to a wide range of cell samples, including single micromanipulated cells (6,7,9,10), antibody fractionated populations (11), and fixed tissues (12). The details of the method
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
are based on a genomic DNA subtraction protocol utilizing biotin addition and xvorganic extraction (13), and the final enriched probes are used to screen full-length cDNA libraries. The advantages of this procedure over the related method of differential display are: the end products are full-length cDNAs, all differences between the starting samples are present after subtraction, and the flexibility of the initial PolyAPCR procedure enables subtraction to be applied to essentially any situation. The disadvantages of PolyAcDNA subtraction are that it can only be applied to relatively few samples, and any clone containing repetitive sequences is likely to be lost.
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