Titlebook: Differential-Display Reverse Transcription-PCR (DDRT-PCR); Sergio Colonna-Romano,Antonella Leone,Bruno Maresc Book 1998 Springer-Verlag Be

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Differential Display,26 upstream random primers (H-APs) requires a total of 312 PCR reactions. Less than 3.5 μg total RNA is needed, and the amplified cDNAs can be analyzed on 13 sequencing gels, each consisting of 24 lanes.

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Cloning of Amplified cDNA Fragments,wever, if smearing or multiple bands appear in PCR products, it is necessary to purify the reamplified cDNA prior to subcloning. Many commercial kits (l.e., QIAquick Gel extraction Kit, Qiagen) allow rapid purification in only a few steps and are relatively inexpensive.

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Confirmation of Differential Expression of Cloned cDNA Fragments,represents a gene differentially expressed in the system under study. Slot blot analysis may also be used for preliminary screening of a large number of putative positive cDNAs, but results need to be reconfirmed on Northern blot.

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Overview,in biology today. Until recently, the methods most widely used to distinguish mRNAs expressed in different cell types or in cells grown under different conditions were techniques based on subtractive (Zimmerman et al. 1980) or differential hybridization (St. John et al. 1979). Although several genes

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Size Separation of cDNA Fragments,mplified cDNAs (Fig. 1). Nondenaturing gels can also be used to display differentially expressed bands. However, nondenaturing gels have not replaced denaturing ones, since both incompletely annealed single-stranded cDNAs and heteroduplexes (produced by errors of Taq polymerase) from the same gene m

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Reamplification of Eluted cDNAs,μl of the eluted cDNA is used as template for the reamplification step in the presence of the same pair of primers used for differential display. The annealing temperatures and the number of cycles are the same as in the first amplification step, but the concentration of dNTPs should be higher (20 μ

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Cloning of Amplified cDNA Fragments,wever, if smearing or multiple bands appear in PCR products, it is necessary to purify the reamplified cDNA prior to subcloning. Many commercial kits (l.e., QIAquick Gel extraction Kit, Qiagen) allow rapid purification in only a few steps and are relatively inexpensive.