基因表达差异显示实验

The usefulness of the purine and pyrimidine salvage pathways in the study of the mechanisms of mutation and in the selection of cell lines stably transformed by vectors expressing these genes is well documented. Unfortunately, many investigators are deterred from selecting new host stra ...

The Sinuclease protection procedure allows the precise definition of the beginning and end of gene transcripts as well as the position of intron/ exon boundaries in a gene. Alternatively, when these parameters are already known, the technique can be used to quantify transcript levels in a vari ...

In the analysis of gene expression, the steady-state level of RNA transcripts is one of the most convenient parameters used to monitor the activity of an endogenous or introduced gene in cell lines and tissues. A variety of methods, such as Sl hybridization (Chapter 21, this vol), RNase protection (C ...

Primer extension is a relatively quick and convenient means by which transcription from a gene transfected into tissue-culture cells can be monitored. The technique can be used to accurately determine the site of transcription initiation or to quantify the amount of cap-site-specific m ...

The level of expression of a given gene in a particular cell type is reflected by the concentration of the resultant messenger RNA. This is subject to regulation during a number of processes, including synthesis, processing, export from nucleus to cytoplasm, and degradation. Clearly, the rate of ...

Antibodies, in general, provide the most sensitive and specific methods for detecting the protein products of genes. Immunoperoxidase techniques described here detect 103-105 molecules/cell (depending on whether the protein is dispersed within the cell or concentrated at high d ...

Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. T ...

Because of their unique transmission properties and male specificity, markers located on the nonrecombining region of the Y chromosome (NRY) have become an important tool in forensic investigation. In the past few years, more than 50 polymorphic Y chromosome-specific short tandem rep ...

In this chapter we review and compare the online single nucleotide polymorphism databases that are now available as research tools. We give an outline of the search strategies that can be used to ensure the most appropriate loci for forensic applications are chosen.

Here, a single nucleotide polymorphism typing methodology is described based on polymerase chain reaction monitoring, in real time, of fluorescently labeled amplified products using the LightCycler. The main advantages of the system are the time required for the analysis (about 20 mi ...

Single nucleotide polymorphisms (SNPs) are emerging as new markers of interest to the forensic community because of their abundance in the human genome, their low mutation rate, the opportunity they present of analyzing smaller fragments of deoxyribonucleic acid (DNA) than with short t ...

In the multiple-color primed in situ labeling (multi-PRINS) technique, using ddNTPs between two PRINS reactions can block the free 3’-end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction, using it as a primer to perform spurious elongation at nondesired sites. ...

Both primed in situ labeling (PRINS) and peptide nucleic acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures present several features (specificity, discriminating abili ...

Primed in situ labeling (PRINS) has proven to be an attractive alternative to fluorescence in situ hybridization for in situ DNA labeling and for being combined on the same slide with others methods. For the aim of a study on the asymmetrical segregation of the chromosomes of the megakaryocytes dur ...

Primed in situ labeling (PRINS) can be used to localize single-copy genes and unique sequences. Using a modified PRINS method that incorporates multiple primers for the same sequence, single-step annealing and extension, anti-Taq DNA polymerase antibody, and stringent washing, we loc ...

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for ini ...

Based on the direct in situ mixing of the colors of different fluorochromes (fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Cascade Blue) incorporated in sequential primed in situ labeling (PRINS) reactions, a new multicolor PRINS procedure is described, ...

The chromosomal regions 13q14 and 17p13 often are found rearranged in hematopoietic tumors in humans, but the rearrangements can be subtle and can escape detection on gross cytogenetic analysis. For example, submicroscopic perturbations of the RB1 and p53 tumor suppressor genes, loca ...

This chapter provides detailed methods and material descriptions of in situ polymerase chain reaction protocols. It describes all the essential components of the technique, various protocols suitable for different kinds of tissues and cell preparations, and also gives various in ...

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are caused in most cases by deletions of the DMD gene. These rearrangements are detectable in affected boys and men by a simple multiplex polymerase chain reaction approach. However, this technique is not able to disclose DMD de ...