Part A Construction And Screening Of A Subtracted cDna Library

Construction of a subtracted cDNA library can allow identification of specific cDNA clones corresponding to a specific class of mRNAs expressed in one cell or tissue type but not in another type. For instance, treatment of cultured cells or organisms with an important chemical, drug, alcohol or hormone may induce or repress the expression of certain genes. In some cases, some novel genes encoding new mRNAs and proteins can be identified corresponding to a specific treatment, which may provide fundamental information about the action or mechanisms of the treatment. Once a novel gene is isolated, it allows opening a broad research area concerning the treatment with promising results. Additionally, a subtracted cDNA library consists of enriched cDNAs that provide a powerful tool to isolate some rare cDNAs transcribed from rare mRNAs, which may be difficult to be identified via screening of a complete cDNA library.1,2

The general procedure is outlined in Figure 3.2. The first step is to isolate total RNAs or mRNAs and to make the first-strand cDNAs representing all of the mRNAs expressed in each of two cell or tissue types of interest. The single-stranded cDNAs

. yy — _ , . I J^Vv Characterization of the isolated cDNAs of interest in type A or B

Screening and isolation of the cDNA clones of interest in type A or B The first strand cDNAs^ ▲ ▲

J Ligation to adaptors/linkers and then to vectors (e.g., Xgt10)

FIGURE 3.2 Scheme of construction and screening of a subtracted cDNA library.

Copyright 2004 by CRC Press LLC

in one cell or tissue type are subjected to hybridization with total mRNAs from another type. For example, in cell or tissue type A, any cDNAs that represent sequences expressed in type B will form DNA/mRNA hybrids with the corresponding mRNAs in cell or tissue type B, or vice versa. Therefore, the unhybridized cDNAs are specific in type A, but not in type B, or vice versa. Single-stranded cDNAs and cDNA/mRNA hybrids can be separated by chromatography on hydroxyapatite columns. The unhybridized cDNAs are then used to synthesize double-stranded (ds) cDNAs, which are then ligated to appropriate adaptors and vectors and utilized to construct a subtracted cDNA library in which the sequences specific to one cell or tissue type are greatly enriched. Compared with a complete cDNA library, a subtracted cDNA library contains an enriched set of cDNA clones, which simplifies the screening procedures, using much smaller numbers of pfu in the primary screening. As a result, the purification process of the cDNA of interest can be speeded up a great deal.1

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