Subtraction Step Research Articles

Streamlined representational difference analysis for comprehensive studies of numerous genomes

Representational Difference Analysis (RDA) is a powerful but also quite complex and difficult method to reveal the variable genes among highly similar genomes or transcriptomes. This report is on the substantial simplification of RDA for microdiversity studies on Bacillus isolates. Following improvements are demonstrated: the average fragment size has been pushed up (∼700 bp) by using nucleotide-biased restriction enzymes, in this case the “high-GC” enzyme HhaI for low GC Gram-positive Bacillus. A PCR-based production of the DRIVER has been applied to avoid subculturing of strains and repeated large-scale DNA extraction. Tedious estimation and adjustment of DNA concentrations for the subtractive hybridization step were by-passed by coupling DRIVER and TESTER production. The problem of linear amplification of TESTER/DRIVER hybrids has been eliminated by applying two different adapters, one for the TESTER but also another one for the DRIVER. Hence, only one RDA round is required to achieve clear bands without background. After doing this streamlined DRIVER and TESTER production, the RDA analysis may now be performed in one night and day. This simplification will allow to scale up the number of genomes under investigation.

PCR‐Based Subtractive cDNA Cloning

Subtractive cloning is a powerful technique that allows isolation and cloning of mRNAs differentially expressed in two cell populations. In the generalized subtraction scheme the cell types to be compared are the [+] or tracer cells and the [-] or driver cells, where mRNAs expressed in the tracer and not the driver are isolated. Briefly, tracer nucleic acid (cDNA or mRNA) from one cell population is allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences common to both cell populations. Hybrids between the tracer and driver, and all driver sequences, are removed in the subtraction step. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. The method described in this unit uses double-stranded cDNA (ds cDNA) as both tracer and driver. Reciprocal subtractions are performed between two cell populations, A and B: that is, genes preferentially expressed in A more than in B are isolated, as are genes expressed preferentially in B more than in A. The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction. The progress of subtraction is monitored by slot blot hybridization. Differentially expressed cDNA sequences are used to construct a subtracted cDNA library.

A spatially varying Compton scatter correction for SPECT utilizing theintegral Klein-Nishina cross section

An algorithm correcting for the fraction of scattered events in SPECT andplanar imaging is proposed. The algorithm utilizes a pixel-based multi-channelanalyser for data acquisition. The method was designed to operate on a locallevel by three subtraction steps: (a) Subtracting a modified Klein-Nishinasingle scatter distribution, pixel by pixel, from the events obtainedexperimentally in the upper half of the photo-peak window. (b) Subtracting amirrored distribution of the unscattered events hence obtained from that oftotal events in the lower half of the window, thus giving the scatterdistribution in this part of the window. (c) Subtracting the sum of the scatterdistributions in both window halves from the corresponding sum of total eventsin order to obtain the unscattered photons within the photo-peak window.The accuracy of the method was validated experimentally, using a new rCBFphantom allowing for imaging in matter corresponding to soft tissue andapproximately in air, respectively. After correction for photon scattering andattenuation, the regional difference in SPECT values in soft tissue equivalentmatter and in low-density matter (simulating air) was only 1.5±7.2%(mean ± 1 SD), thus indicating a high accuracy of the correctionmethod. Provided that an accurate and stable pixel peak-alignment routine isavailable, the method can be applied using a minimum of three windows.

Assessment of Subtractive Hybridization to Select Species and Subspecies Specific DNA Fragments for the Identification of Xylophilus ampelinus by Polymerase Chain Reaction (PCR)

Eighteen Bsp143I digested DNA fragments specific to Xylophilus ampelinus were cloned from a library enriched for X. ampelinus obtained after a subtractive hybridization step. It was also possible to clone specific DNA sequences directly after DNA digestion with Bsp143I probably because X. ampelinus is a unique bacterium. Nucleotidic sequences of four cloned specific fragments were determined. They did not share any homology with other DNA sequences in the EMBL/GeneBank database. Four primer sets were designed and tested for specificity to X. ampelinus. One primer set (Xamp 1.27) was a good candidate for a species-specific reagent in a procedure of identification of X. ampelinus using PCR. One primer set detected only Greek strains isolated from Vitis vinifera cv. Sultana. Genetic diversity within the X. ampelinus species can be used in further epidemiological studies on the bacterial necrosis of grapevine.

20] Suppression subtractive hybridization: A versatile method for identifying differentially expressed genes

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

Differential Subtraction Display: A Unified Approach for Isolation of cDNAs from Differentially Expressed Genes

We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/−) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Out-of-plane photon compensation for 3-D SPECT image reconstruction with generalized matrix inverses

A computationally efficient 3-D image reconstruction method which compensates for detected out-of-plane photons has been developed for SPECT image reconstruction with generalized matrix inverses (GMI). Fully 3-D image reconstruction is approximated by a series of coupled 2-D image reconstructions for projection data acquired with parallel hole collimators, significantly reducing computer memory requirements. In this method, projection data are compensated for detected scattered photons using dual energy window scatter subtraction (Step 1). An initial source activity estimate in each transverse plane is then made using line source response functions in the system matrix (Step 2). With these activity estimates the contributions of out-of-plane unscattered photons are modeled and subtracted from the projection data, and an updated source activity estimate is computed (Step 3). For noise-free projection data from a Monte Carlo simulated myocardial perfusion study, lesion contrast increases and activity spillover from the myocardium into the adjacent cardiac blood pool is reduced. For projection data with Poisson noise, activity estimates from Step 3 are inferior to those from Step 2. One reason is that the relative noise level of the projection data is substantially increased by subtracting the estimated out-of-plane contribution. The Step 3 activity estimation is also sensitive to the source activity distribution used for out-of-plane unscattered photon compensation. Though the out-of-plane compensation technique of Step 3 provides some benefit for noise-free projection data, this study suggests that it may not be well-suited for practical application to count-limited clinical SPECT studies.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Optimization of subtractive hybridization in construction of subtractive cDNA libraries

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C ot values.

Development and application of an elisa technique for the detection of antibody to avian encephalomyelitis viruses

A rapid sensitive enzyme-linked immunosorbent assay for the detection of antibody to avian encephalomyelitis viruses (AEVs) in chickens using purified antigen is described. The procedure differed from others which have been described for AEV, in that it involved a negative antigen subtraction step which accounted for the variable adhesiveness of chicken sera to plastic surfaces. The procedure was reproducible (between-assay coefficient of variation 8.95 per cent) and a good correlation was observed with results obtained by neutralisation index tests (r = 0.91, P less than 0.1). The assay detects only AEV-specific antibody and allows monitoring of the spread of AEV in flocks.

Repetitive spectral subtraction method for the spectrophotometric determination of rare earth elements

A new multiwavelength data-analysis method for the determination of multicomponent mixtures, the repetitive spectral subtraction method (RSSM), is proposed. In RSSM, spectral data are obtained at evenly spaced wavelengths in the range of interest, and the analysis of the spectrum is performed as repetitive determination-subtraction cycle. In the determination step, the series of data in the narrow wavelength range is used, which is specified as to include the most characteristic or the largest peak for each component. Assuming a linear background, the target component, which can be a small group of components, is simultaneously determined together with co-existing components. In the next subtraction step, the calculated contribution of the component is subtracted from the spectral data in the whole measured wavelength range, and the component is assigned as determined and is excluded from the following analysis. This determination-subtraction cycle is repeated until all the components are determined.

Sharing Teaching Ideas: A “No Borrow” Subtraction Algorithm

If an algorithm is to be truly of low stress, then it most certainly must address itself to the immediate problems of failure. The low-stress subtraction algorithm presented in this article is a result of personal observation of the frustrations and difficulties encountered by my ninth-grade mathematics students. The algorithm eliminates the traditional borrowing process. Students may “rest" in the middle of the problem and do not have to perform all the subtraction steps in a prescribed manner from right to left.

The least-squares line and plane and the analysis of palaeomagnetic data

Summary The classic, multivariate technique of principal component analysis can be used to find and estimate the directions of lines and planes of best least-squares fit along the demagnetization path of a palaeomagnetic specimen, thereby replacing vector subtraction, remagnetization circles and difference vector paths with one procedure. Eigenvalues from the analysis are the variance of the data along each principal axis, and provide a relative measure of collinearity or coplanarity which may be used to define a general palaeomagnetic precision index. Demagnetization planes found with principal component analysis may be used in place of difference vector paths for locating Hoffman—Day directions, avoiding unnecessary vector subtraction and intensity truncation steps. Two methods are discussed for jointly estimating an average remanence direction from demagnetization lines and planes.

A method of incoherent optical-image processing using synthetic holograms*

Under coherent illumination, the interference between signal and noise produces an amplification of the noise. That is why image processing by optical methods may be done better with incoherent light than with coherent light. A method is described where image deblurring is performed in spatially incoherent illumination. The two main features are an additional step of illuminance subtraction to simulate negative intensities, and the use of computer-generated holograms of a low number of cells.

Subtraction steps

While games that provide practice in subtraction are easily located, those that also build an understanding of the inverse relationship between addition and subtraction are not easily found. A few years ago we developed an activity the children called “Subtraction Steps” that seemed to do just that. It was fun. It provided practice and yet strengthened the children's understanding of the inverse operations of addition and subtraction. And, as an added dividend, children soon found they could easily make their own “steps” to contribute to the arithmetic table.