实验11 cDNA的合成及RT.pptVIP

* 实验13 cDNA的合成及RT-PCR法研究基因的表达 RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. Two-step procedures In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR. Advantages of this approach: This method is useful for experiments where multiple transcripts have to be analyzed from the same RT reaction or for specific applications such as Differential Display Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the appropriate enzymes are used). A two-step procedure has the following advantages Optimizes reaction conditions. The two-step format allows both reverse transcription and PCR to be performed under optimal conditions to ensure efficient and accurate amplification. Provides flexibility. Two-step procedures allow the product of a single cDNA synthesis reaction to be used for analysis of multiple transcripts. This flexibility is valuable for such specialized applications as rapid amplification of cDNA ends (RACE) and Differential Display Reverse Transcription (DDRT). Amplifies long sequences. With the right combination of reverse transcriptase and thermostable DNA polymerase, two-step