Autoradiography polymerase chain reaction

Fig. 5. (Opposite page) Polymerase chain reaction (PCR)-based expression of cDNA information using conventional method (A) and a new strategy using split-type primers (B). a. Design of the split-type primers for the introduction of the required UTRs into cDNA sequences, b and c. Expected PCR-generated DNAs and mRNA, respectively, d. PCR-generated DNA. e. A 10-pL aliquot from aPCR sample was used forthe 100-pL transcription reaction, and transcripts were analyzed, f. All of the transcript was used for batch-mode translation (50 pL, 4 h), and products were analyzed by autoradiography. (From ref. 36a.)...

Detection with a microchip is primarily through LIF, since this is eashy implemented with the planar configuration of the microchip (Figure 5-10). Limits of detection for fluorescein-like fluors have been easily demonstrated at the 10 M level and pushed as low as 10 M—a mass detection limit of a few hundred molecules. This allows for detection, for example, of polymerase chain reaction (PCR)-amplified DNA fragments at a level that competes with P-autoradiography jffom Southern blots. Typical microchip separation times are around 50 to 200 seconds. 

Fig. 17.7. The Sanger method. (A). A reaction mixtures contain one of the dideoxynucleotides, such as ddATP, and some of the normal nucleotide, dATP, which compete for incorporation into the growing polypeptide chain. When a T is encountered on the template strand (position 10), some of the molecules will incorporate a ddATP, and the chain will be terminated. Those that incorporate a normal dATP will continue growing until position 15 is reached, where they will incorporate either a ddATP or the normal dATP. Only those that incorporate a dATP will continue growing to position 17. Thus, strands of different length from the 5 end are produced, corresponding to the position of a T in the template strand. (B). DNA sequencing by the dideoxynu-cleotide method. Four tubes are used. Each one contains DNA polymerase, a DNA template hybridized to a primer, plus dATP, dGTP, dCTP, and dTTP. Either the primer or the nucleotides must have a radioactive label, so bands can be visualized on the gel by autoradiography. Only one of the four dideoxyribonucleotides (ddNTPs) is added to each tube. Termination of synthesis occurs where the ddNTP is incorporated into the growing chain. The template is complementary to the sequence of the newly synthesized strand.

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