PCRRT-PCR指南（国外英文资料）.docVIP

PCRRT-PCR指南 5and 3 RACE RACE (cDNA, Rapid, Ampliphication, of, Ends, cDNA) is a method of obtaining transcripts of unknown sequences of 5or 3. Do not use as the normal RT-PCR two sequence specific primers, RACE uses a sequence specific primers and mRNA or poly (A) tail (3RACE) or to cDNA at the end of the poly homopolymer (5 RACE) (Figure 11). RACE has been used to amplify and clone rare mRNA. RACE products can be used for cloning, direct sequencing, preparing probes, or linking together to obtain full length cDNA. One of the ways to connect 5and 3 RACE products is to design new primers for amplification of full-length cDNA using sequence information obtained by 5and 3 RACE. Full length cDNA clones are obtained by high fidelity amplification of long sequences using RNaseH- reverse transcriptase and high fidelity heat stable polymerase. 5, RACE, step outline, first strand primer, GSP1, and mRNA annealing Use SuperScript II to translate mRNA into cDNA Degradation of RNA using RNase mixtures Purify cDNA using GlassMAX Spin Cartridge Purified tails of cDNA were purified using dCTP and TdT CDNA was amplified with degenerate anchor primers and nested GSP2 PCR with dC tail Preliminary PCR products were re amplified using AUAP, UAP, and nested GSP. Figure 11. 5RACE step outline 5RACE is more challenging and less specific than RT-PCR because only one primer is genetically specific. The product of 5RACE may be a single product, multiple products, or even a continuous band that is not distinguishable. The quality of the results depends on the specificity of the mRNA used to synthesize the first and amplified GSP, the specificity of the anchor primers used, the complexity and abundance of the target, and the length of the product. Amplified using nested primers (up to three nested amplification), and in successive rounds of length selective amplification of PCR product as template can increase the specificity of 5RACE (see Chapter fifth, nested PCR). Increasing the reverse ther