Subtractive library

Many research projects that aim to establish the identity of differentially expressed genes from two or more samples would not necessarily benefit from the microarray approach compared to more conventional methods such as subtractive library screening or differential display. Initial considerations as to the suitability of this technique to a particular application include whether more than two samples are being compared, how many differences might be expected, and what proportion of the total number of transcripts from the target tissue are known or have been cloned. In addition, the practical issues of availability of array robotics and analysis hardware and technical expertise must all be considered. 

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. 

Spectral searching and stripping in the analysis of a mixture of mannitol and cocaine hydrochloride, (a) IR spectrum for the mixture (b) Library IR spectrum of mannitol (c) Result of subtracting mannitol s IR spectrum from that of the mixture ... 

To record a mass spectrum it is necessary to introduce a sample into the ion source of a mass spectrometer, to ionize sample molecules (to obtain positive or negative ions), to separate these ions according to their mass-to-charge ratio (m/z) and to record the quantity of ions of each m/z. A computer controls all the operations and helps to process the data. It makes it possible to get any format of a spectrum, to achieve subtraction or averaging of spectra, and to carry out a library search using spectral libraries. A principal scheme of a mass spectrometer is represented in Fig. 5.2. To resolve more complex tasks (e.g., direct analysis of a mixture) tandem mass spectrometry (see below and Chapter 3) may be applied. 

FJciureJyT. (a) Mass spectrum at the top of the chromatographic peak (b) background spectrum (c) analyte spectrum after background subtraction (d) library spectrum of hexachlorobiphenyl (score 99%). 

Before the comparison of the recorded spectra with those in a library it is worth estimating its quality and making possible improvements. For example, working in GC/MS mode it is always helpful to subtract the background. 

Fig. 21.11. Mass spectra of the unknown off-flavor compound after spectral subtraction from the co-eluting peak and the matching spectrum from the NIST library. (Redrawn/redrawn from J. Chromatogr., 351, R.A. Sanders, and T.R. Morsch, Ion profiling approach to detailed mixture comparison. Application to a polypropylene off-odor problem, 525-531, Copyright (1986) with permission from Elsevier.)...

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