Since the cytosine addition is cap-dependent, the anchor primer is only appended to the 3′ ends of full-length cDNA. Then an anchor primer with multiple guanine residues (poly(G)) is added to the reaction mixture and primarily annealed to the exposed poly(C) in the 3′ ends of first-strand cDNA, introducing an adaptor to the first-strand cDNA terminus. The Cap-switching RACE (Cap finder) is performed by moloney murine leukemia virus (MMLV) reverse transcriptase to add extra 2-4 cytosines to the 3′ ends of newly synthesized first-strand cDNAs after reaching the cap structure of the 5′ end of mRNAs 5, 6. The method has been applied and developed in cRACE and RLM-RACE 4, 9. However, when the mRNAs are incompletely transcribed for lacking the 5′ end, the above progresses are not performed therefore the full-length cDNAs are only amplified using these anchored and SPR primers 7, 8. The full-length mRNAs have methylated ‘G’ caps at their termini, when treated with shrimp alkaline phosphatase (SAP) to remove the phosphate and then T4 RNA ligase is available to ligate the anchor RNA adaptor to the 5′ ends.
In new RACE, an anchored RNA adaptor is ligated to the 5′ ends of mRNA before conducting the reverse transcription (RT) reaction. Nowadays, several RACE methods including new RACE and Cap-switching RACE have been developed, especially the 5′ RACE for the difficulty in operation 3, 4, 5, 6. This method is complex in operation and low in productivity. The Outer-R and Inner-R primers are again used with the 5′ sequence-specific reverse primers (SPRs) to obtain the 5′ ends of cDNA 1, 2. While a poly(A) tail is tailed to the 3′ ends of first-strand cDNA by terminal transferase and then reversed to second-strand cDNAs by anchor-sequence tagged oligo(dT) primers described above. In classic RACE, an anchor-sequence tagged oligo(dT) primer is used to reverse mRNA into first-strand cDNA, then the 3′ end sequences of mRNAs are obtained by nest-PCR by the 3′ sequence-specific forward primers together with Outer-R and Inner-R primers (corresponding to the anchor sequence). Although the NGS technique is produced and developed in the 21 st century, researchers still use the PCR technique known as rapid amplification of cDNA ends (RACE) to obtain the full-length cDNA sequences 1. However, the transcriptomes are always incomplete in length, especially the ends of genes, while the complete genome sequencing is only conducted in some limited species due to the complexity and high cost. The transcriptomes of increasing number of species have been sequenced by next generation sequencing (NGS) technique. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. The putative 5′ ends are further validated by primers corresponding to these predicted sites in cDNAs. The 5′ end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species and screening the relative locations of common characteristic cis-elements in silico on promoter. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5′ and promoter sequences. For the amplification of 5′ ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5′ sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database.
The amplification of 3′ ends of cDNA is performed according to the modified classic 3′ RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed.
A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study.
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