已知一个cDNA3’端的部分序列,请设计实验流程得到该基因的全长cDNA。

1，根据已知序列设计三条巢式引物，进行5’RACE，拿到5’序列；
2，同时设计巢氏引物，进行3’RACE拿到3’序列；
3，序列拼接；
4，设计引物扩增全长序列

The first step in RACE is to use a reverse transcription-polymerase chain reaction (RT-PCR) to produce a cDNA copy of a region of the RNA transcript. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript. The copied region is bounded by the known sequence, and either the 5' or 3' end.

The protocols for 5' or 3' RACE differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription) reaction using an anti-sense (reverse) oligonucleotide primer that recognizes a known sequence in the gene of interest; the primer is called a gene specific primer (GSP), and it copies the mRNA template in the 3' to the 5' direction to generate a specific single-stranded cDNA product. Following cDNA synthesis, the enzyme terminal deoxynuc