Digoxigenin colorimetric detection

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

PCR can be used to introduce labels that can then be used for detection. The ability to add to the 5 end of the primers sequences not complementary to the target template, which then becomes incorporated into the double-stranded PCR product, allows the introduction of labels. Thus, the addition of biotin to the 5 end of the primer allows detection of hybridized PCR product with streptavidin or avidin-enzyme conjugates (B4). Other labels such as digoxigenin can be added to the 5 end of the primer, amplified, and detected either colorimetrically or by chemiluminescence (F3). 

Chapters 28 and 29 describe the use of nonradioactive probes to detect gene transcripts in thin sections in situ. Chapter 30 describes the use of nonradioactive probes on whole embryo mounts. The probes used in this section are digoxigenin-labeled RNA probes, and the detection is colorimetric, revealing the cell and tissue types that are expressing particular genes. 

This chapter describes the use of digoxigenin-labeled probes to detect mRNA transcripts in tissue sections. The sites of hybridization of the probe are visualized using an antidigoxigenin antibody alkaline phosphatase conjugate, and a colorimetric reaction. 

There are a number of different probes available, including detection probes, which are labelled with a fluorescence marker or, for example, digoxigenin for a further linkage with an antibody-enzyme complex and then a later colorimetric reaction with a chromogenic substrate (such as nitro blue tetrazolium for alkaline phophatase) (Helentjaris McCreery, 1996 Kempf et al., 2000), Capture probes are used to bind the target sequence (RNA or DNA) to a plate or another surface. In most cases the probes are labelled with biotin to react with avidin, which is coated on a plate (Riley, Marshall, Coleman, 1986). 

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