Protein-nucleic acid interactions have been studied by density shift gradients, affinity columns, and gel retardation. Some of these methods are tedious or have severe limitations in quantitative analysis. Filter binding assays are fast, sensitive, and suitable for physical chem ...

Type I topoisomerases catalyze the reversible nicking of duplex DNA (for review, see ref. 1). In the nicked state, the enzyme is attached to the DNA by way of a phosphodiester bond between a tyrosine residue in the protein and the end of the broken strand. The polarity of attachment depends on the source of the en ...

Serial analysis of gene expression (SAGE™) is a patented, large-scale mRNA-profiling technology that produces comprehensive, quantitative, and reproducible gene expression profiles (originally described in refs. 1 and 2). Unlike the alternative technologies of differenti ...

Much of modern genetics is based on analysis of DNA sequence. Therefore, there is great pressure to scale up sequence analysis while decreasing its cost. The most promising platforms are based on the use of oligonucleotide arrays (DNA chips), which perform many analyses in parallel (1). Arrays co ...

RNA arbitrarily primed polymerase chain reaction (RAP-PCR) has been used extensively to identify differentially regulated genes (1–6). The RAP-PCR method begins with conversion of RNA into cDNA, followed by arbitrarily primed PCR. The technique uses arbitrarily primed PCR (7–11) to am ...

Differential display of mRNA via polymerase chain reaction (DD-PCR) has become a powerful procedure for the quantitative detection of differentially expressed genes in distinct cell populations (1–4).

AU-rich elements (AREs) are found in 3′ untranslated regions (3′ UTR) of many highly unstable mRNAs for mammalian early-response genes. The minimal AU sequence core within the ARE is the heptamer WAUUUAW, although from a functional point of view, several pentanucleotides clustered in close p ...

Since the completion of the human genome-sequencing project, scientists are now able to read the code of all human genes stored on the 46 chromosomes of the human genetic library. However, we are far from reaching an understanding of the functional relationships existing between more than a tiny f ...

Real-time quantitative polymerase chain reaction (PCR) methods (1) can be further optimized for various purposes by performing quantitative PCR for multiple RNA species in one sample (2). Advantages of this method not only include the elimination of differences in reaction mix volumes ...

Precise and accurate determination of mRNA expression levels in tissues and model systems is a central methodology in a wide range of research applications. Expression of many genes is currently assessed by northern blotting, RNAse protection assays, Serial Analysis of Gene Expressi ...

The use of quantitative polymerase chain reaction (qPCR) to detect RNA viruses has become increasingly important as a prognostic marker and in patient management, for example, in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Drug therapies can be monitored by r ...

Chromosomal aberrations, such as translocations or inversions, described for a growing number of malignancies, are now widely used to detect tumor cells by polymerase chain reaction (PCR). However, in multiple myeloma (MM), no such ubiquitous PCR marker exists. Therefore, other means ha ...

The nature of the polymerase chain reaction (PCR) process lends itself well to qualitative determinations. It transforms very small quantities of analyte into the realms of bucket chemistry, allowing specific gene portions to be directly visualized with ethidium bromide and ultrav ...

In this chapter, we detail protocols of long polymerase chain reaction (PCR) and long RT-PCR, which we have found to be versatile, sensitive, and straightforward to optimize. We have used these protocols with success on several different templates, including lambda phage DNA, HAV, HBV, HCV (1), to ...

The polymerase chain reaction (PCR) has become an essential and ubiquitous tool for biological research and laboratory diagnostic applications. Until recently, reliable and sensitive amplification of large templates (several kb) was difficult to achieve. However, in 1994, an imp ...

Nucleic acids used for polymerase chain reaction (PCR) assays usually are extracted by the phenol-chloroform method or an alternative rapid purification. The acid-guanidinium thiocyanate-phenol-chloroform method for RNA extraction and proteinase K digestion-phenol-c ...

Single nucleotide polymorphisms (SNPs) are single base differences in genomic DNA (1). These single-base mutations, estimated to occur every 1000 bases, are thought to represent the most common form of genetic variation in the human genome (2). Several million SNPs have been identified (3). H ...

The study of genetic polymorphism among diverse populations of organisms is a complex task. However, this can be accomplished by using newer tools, such as randomly amplified polymorphic DNA (RAPD). RAPD is a polymerase chain reaction (PCR) technique that relies on the generation of amplifi ...

Hemophilia A is an X-linked disorder caused by mutations in the factor VIII gene. Around 50% of all patients with severe hemophilia A share a common mutation. This intron 22 inversion results from homologous recombination of a sequence within intron 22 of the factor VIII gene and identical sequence ...

There are several methods now in widespread use for detecting and characterizing specific RNA targets. These methods include in situ hybridization, Northern blotting, dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase chain reaction (RT-PCR). ...