Subtractive hybridization, cloning

Kim M, Kim S, Kim S, Kim B-D (2001) Isolation of cDNA clones differentiaUy accumulated in the placenta of pungent pepper by suppression subtractive hybridization. Mol CeUs 11 ... 

Subtractive hybridization is a method that enables one to quickly obtain large numbers of cDNAs that are differentially expressed between control and experimental conditions25,31,34 50 56 57 83 106. To perform this procedure, one must first isolate poly-A + RNA from both treated and control tissues. The poly-A + RNA is then reverse transcribed into cDNAs and then, by a series of manipulations, the two fractions are hybridized together. cDNAs present in both fractions form duplexes and are removed from consideration. cDNAs that do not hybridize because they are preferentially expressed in either the control or the treated samples can then be PCR amplified, cloned, and subsequently sequenced. The method is normally performed to obtain both up- and down-regulated genes. 

Sangrador-Vegas, A., J.B. Lennington and T. Smith. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization. Cytokine 17 66-70, 2002. 

In a mammalian cell, a large number (about 10 000) of mRNAs have a low abundance, i.e., a few copies per cell. Many strategies have been designed to enrich for specific mRNAs or to recognize certain clones (Section 2.5.2.2) otherwise many clones would have to be analyzed (Table 10.1). Subtractive hybridization depletes the sample of nonspecific mRNA in order to enrich for the mRNA sought. It is particularly suitable for isolating tissue-specific or developmental regulated sequences or clones derived from mRNA induced by external factors. 

High ratio subtractive hybridization by cloning inhibition In the method described by Klickstein (1987), ( + ) cDNA with certain restriction sites (e.g., EcoRI) and a fragmented (-) cDNA library lacking these restriction sites are mixed in a proportion of 1 50, denatured and hybridized so that only (-I-) DNA unhybridized with ( -) DNA can be cloned in EcoRl-digested vectors (Fig. 12.1(1)). 

Cloning of BR-induced genes in soybean by subtractive hybridization. 

An efficient and rapid subtraction hybridization technique (RaSH) allows the identification and cloning of differentially expressed genes . ... 

LUO D, HU w, CHEN s, XIAO Y, SUN Y and ZHU z (2009) Identification of differentially expressed genes between cloned and zygote-developing zebrafish (Danio rerio) embryos at the dome stage using suppression subtractive hybridization. Biol Reprod, 80, 674-684. 

A set of 60 cDNAs were isolated by subtractive hybridization of Nicotiana tabacum roots before and after removal of the flowers and young leaves, a process known as topping [32], Topping is known to increase the amount of nicotine and other related alkaloids in the plant. A number of the subtracted cDNAs that were unique to the topped plants corresponded to known alkaloid biosynthetic enzymes. The functions of additional cDNAs observed in the subtracted library were not clear and remain under investigation. Further study of these new clones may provide insights into alkaloid natural product biosynthesis in N. tabacum. 

A 30-fold enrichment is usually obtained allowing < 6 transcripts per cell to be identified. It is essential to complete the hybridization reaction as much as possible without having a large excess of driver . Clones tend to be small with this method. A major drawback is that variation between (-I-) and (-) expression of different genes may reduce the efficiency of low ratio hybridization subtraction. 

The simplest expression arrays consist of individual cDNA clones manually or robotically spotted onto nitrocellulose or nylon filters. Replicates of the clone arrays are then hybridized sequentially or in parallel with different radioactively labeled target cDNAs, and hybridization to each clone measured by Phosphor Imager or similar technologies (33-36). Our own laboratory used this simple approach to assess the efficacy of protocols to generate subtracted cDNA libraries (37). Filters with thousands of arrayed human or murine genes and the requisite analysis software have been commercially available for the past several years. 

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