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The ability to compare two different nucleic acid libraries has permitted inquiries into the role of differentially expressed genes or deleted/inserted genomic sequences involved in the mechanisms of neoplastic transformation, developmental regulation, physiological processing, pathological disorder, and therapeutic efficacy. Subtractive hybridization between two complementary DNA (cDNA) libraries is a powerful tool for identifying differentially expressed genes. In principle, an excess amount of modified subtracter cDNAs derived from cells of a control group are used to bind with tester messenger RNAs (mRNAs) or cDNAs isolated from the cells of interest. Because the subtracter cDNAs are modified to interfere with the amplification processes of reverse transcription (RT) and/or polymerase chain reaction (PCR), all subtracter-bound tester sequences are degraded and only the differentially expressed genes in the tester can be preserved for RT-PCR amplification. To improve the efficiency of subtractive hybridization, we have developed a chemical modification procedure to generate covalently binding cDNAs as the subtracter to capture the homologous tester sequences. We have also proved that the covalently bound duplex hybrids cannot be separated in PCR and thus are removed from the amplified differential gene sequences. Using the novel principle of covalently hybridized subtraction (CHS), we provide an easy, fast, and effective subtractive hybridization method for understanding the alterations of gene expression and/or chromosomal rearrangement in disordered cells in comparison with normal ones, which may reveal targets for gene therapy, eugenic improvement, pharmaceutical drug design, and investigation of etiological mechanisms. [ABSTRACT FROM AUTHOR]

Microarray technology has revolutionized molecular biology by permitting many hybridization experiments to be performed in parallel. With the size of a glass microscope slide, this tool can carry thousands of DNA fragments in an area smaller than a postage stamp. In this chapter, we will describe microarray chips that host nucleic acid probes, which are the most commonly used type of microarrays. Science in this field is mostly data-driven, where biological hypothesis are generated upon analysis and comparison of a huge amount of potentially meaningful differential data derived from microarray hybridizations. DNA microarray technology is under a constant and rapid evolution. The first paper reporting DNA microarray as a tool for transcript-level analyses has been published in 1995, and that chip had about 1000 Arabidopsis genes. Almost 11 years have passed and advances in miniaturization, robotic, and informatics, as well as the development of alternative approaches to microarray construction have permitted to put more than 250,000 different spots into a single square centimeter. This rapid advance in the microarray field, combined with the falling price of technology and the acquisition of wholegenome sequence information for hundreds of organisms has caused biologists to abandon their home-made equipment in favor of one of an expanding range of commercial platforms now available on the market. However, we are still not able to represent the entire genome of any eukaryotic organism in a unique chip or even to analyze the great complexity of the human transcriptome. Therefore, how to choose and design the best probes to construct DNA microarray chips is a crucial step to the appropriate use of this powerful technique (Figure 1). [ABSTRACT FROM AUTHOR]

Being very useful and informative, many techniques based on nucleic acids hybridization suffer from the cross-annealing of repetitive DNA, presenting in reassociating samples. This "wrong" annealing causes "nonspecific" hybridization of nonorthologous DNA fragments, thus producing chimeric sequences and at the final stage significantly hampering the analysis of the resulting cDNA or genomic libraries. Such chimeras may constitute up to 40-60% of DNA libraries. Importantly, the number of chimerical clones positively correlates with the complexity of hybridizing genomic or cDNA mixtures. The hybridization specificity is a crucial factor determining both the fidelity the efficiency of all hybridizationbased analytical techniques. In this chapter, I review the current attempts to increase the specificity of hybridization at both stages: during nucleic acids reassociation and at the stage of selection of proper hybrids. To this end, approaches based on chemical modifications, improving hybridization kinetics, and improving selection of perfectly matched duplexes, have been developed. [ABSTRACT FROM AUTHOR]

This group of methods is aimed at the identification of single nucleotide-scale differences between the comparing DNA samples. Mutation and polymorphism detection is of increasing importance in the field of molecular genetics because the study of mutations reveals the normal functions of genes, proteins, noncoding RNAs, the causes of many malignancies, and the variability of responses among individuals. A plethora of single nucleotide polymorphisms (SNPs) are not deleterious by themselves, but are linked to phenotypes associated with diseases and drug responses, thus providing a great opportunity for their use in large-scale association and population studies. Millions of SNPs have been identified in recent years. However, this figure seems negligible compared to the real number of SNPs and other mutations presented in the genomes. Many mutation discovery methods quickly and effectively indicate the presence of a mutation in a sample region, but fail to resolve its characterization and localization; another family of methods permits precise mutation mapping, but in a greatly more laborious and expensive way. The group of novel approaches for mutation detection, which combines high performance, cost-efficiency, reliability, and detailed mutation characterization, will be reviewed in this chapter. [ABSTRACT FROM AUTHOR]

Coincidence cloning (CC) is aimed at finding DNA fragments, which are common to the samples under study. The nature of these input DNAs may be genomic or cDNA, cloned or uncloned. The approach is based on cloning identical (or almost identical) nucleotide sequences belonging to different fragmented genomic DNA or cDNA pools, while discarding sequences that are not common to both. Early versions of the CC technique were not very efficient. Their most serious disadvantage was rather low selectivity, so that the resulting libraries of the fragments contained large amounts of sequences unique to one of the two sets of DNA fragments under comparison. To avoid this, Azhikina and colleagues were the first to exploit the technique of selective polymerase chain reaction (PCR) suppression, which strongly increased the efficiency of CC. Another important problem is the "nonspecific" imperfect hybridization between nonorthologous repetitive elements or short sequence-similar sequences, which produces chimeric clones representing an impressive fraction of the libraries (up to 60%, when complex genomic mixtures). An important improvement in this technique comprises treatment of the hybrids with the nucleases, specifically recognizing single-nucleotide mismatches or more extended loop regions. This results in digestion of improperly matched hybrids (primarily chimeras), whereas perfect, nonchimeric heteroduplexes are greatly enriched in the final mixture (up to 96% or more). [ABSTRACT FROM AUTHOR]

Our knowledge on the subject of genetic divergence is ever expanding and encapsulates a wide spectrum of research areas from the study of single nucleotide polymorphism to examination of the complex differences of host-pathogen interactions. The understanding of genetic differences is essential to our ability to address adequately human health issues caused by mono allelic genetic disorders, altered gene expression in cancers, development of drug resistance and the variety of ways organisms respond to infections and the environment. Large number of hybridization-based applications has been developed to query and find such differences. This review introduces a new method for genetic difference screening — the primer extension enrichment reaction (PEER) — presented in the context of similar subtraction-based hybridization methods. PEER is a novel approach to difference screening and is not intended to replace the existing elegant hybridization methods but to expand their scope. PEER is tailored to find unknown targets present in very low copy numbers and in the context of an imperfect genomic match. The PEER method takes advantage of the greater hybridization specificity of shorter oligonucleotides coupled with enzymatic extension specificity. [ABSTRACT FROM AUTHOR]

In a cellular transcriptome, the number of mRNA copies per gene may differ by several orders. In cDNA libraries, performed from mRNA, these proportions are the same. Normalization methods allow us to equalize numbers of gene's copies in the library. Normalized cDNA libraries are used to discover new genes transcribed at relatively low levels or for functional screenings. Here, we observed different cDNA libraries normalization methods, which were based on hybridization (renaturation) of cDNA or DNA, or RNA. Also we described duplex-specific nuclease (DSN) normalization protocol - simple and effective cDNA libraries normalization method. [ABSTRACT FROM AUTHOR]

As originally designed, stem-loop (SL) hybridization probe is a single-stranded oligonucleotide containing a sequence complementary to the target that is flanked by inverted repeats forming self-complementary termini, and harbors a fluorophore-quencher pair at the 5ʹ- and 3ʹ-ends.When the target is missing, these molecules form closed pan handle-like structures in which the fluorophore and quencher are located in a close proximity, due to 5ʹ- and 3ʹ-termini spatial neighborhood. Such a quencher proximity represses fluorescence. In contrast, in the presence of the target, probe forms a complex with it, which spatially separates the fluorophore from the quencher. Once the fluorophore and quencher are dissociated, the fluoresce increases, thus enabling quantitative measuring of signals with the threshold value evidencing presence of the target. Alternatively, SL probe can be fluorescence resonance energy transfer (FRET)-labeled, with the fluorescence shift message being monitored in the latter case. SL probes are currently in use in a number of applications, primarily for specific nucleic acid motif detection and in real-time polymerase chain reaction (PCR). Also, SL probes can be utilized in protein-DNA interaction studies, and even for specific inorganic ion detection. This quickly evolving group of methods gave rise to at least 40 international patents and is cited in ~50 peer-reviewed papers annually. [ABSTRACT FROM AUTHOR]

Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. To our knowledge, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. It is based primarily on a suppression polymerase chain reaction (PCR) technique (described narrowly in Chapter 3) and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs or genomic DNA fragments and simplifies analysis of the subtracted library. SSH technique is applicable to many comparative and functional genetic studies for the identification of disease, developmental, tissuespecific, or other differentially expressed genes, as well as for the recovery of genomic DNA fragments distinguishing the samples under comparison. This chapter provides an insight into SSH practical use and contains detailed protocol for generation of subtracted cDNAs (which is the most frequent SSH application) and differential screening of the resulting subtracted cDNA library. As shown in many examples, the SSH technique may result in over 1000-fold enrichment for rare sequences in a single round of subtractive hybridization. Finally, we discuss the characteristics of cDNA-subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as procedure for rapid identification of truly differentially expressed cDNA clones. [ABSTRACT FROM AUTHOR]

This chapter is devoted to methods based on suppression polymerase chain reaction (PCR) effect (cDNA library construction starting from a small amount of total RNA; suppression subtractive hybridization, SSH; ordered differential display, ODD; Marathon cDNA RACE; genome walking; variants of coincidence cloning, CC; normalization of cDNA libraries; multiplex PCR, mPCR; to in vitro cloning). Taken together, these approaches allow one to analyze complex DNA samples, from searching sequences of interest to determining complete structures of the respective genes. [ABSTRACT FROM AUTHOR]

Several nucleic acids hybridization-based approaches, such as microarray, competitive genomic, and Southern or Northern blot hybridization, have become popular tools for specialists in biochemistry and in biomedicine, and are now in routine use. However, the potential of in-solution nucleic acids hybridization-based experimental techniques seems to be underestimated now. Examples are subtractive hybridization (SH), which allows one to efficiently find differences in genomic DNAs or in cDNA samples; coincidence cloning (CC), which, on the contrary, makes it possible to identify sequences that are present in all the samples under comparison; cDNA normalization, which is used for the smoothing of rare and frequent transcript content in cDNA libraries; and TILLING approach, which has demonstrated its great potential for the reverse genetics studies. Finally, several techniques are aimed at the large-scale recovery of DNA polymorphisms, including single nucleotide polymorphisms (SNPs). This book will focus on the above-mentioned and other recent developments in the area of nucleic acids hybridization, including attempts to improve its specificity. In this introductory chapter, I have tried to briefly characterize the current state of the art in in-solution nucleic acids hybridization techniques, and to define their major principles and applications. The advantages and shortcomings of these techniques will be discussed here. [ABSTRACT FROM AUTHOR]