LncRNAs are transcribed mainly by RNA polymerase II; they are spliced products via canonical genomic splice site motifs, frequently ended with a poly A tail.
On the basis of the collective work presented here, we propose HIV-1 uses RNA structural beacons to direct recruitment of hnRNP A1 to specific genomic splice sites.
Emerging evidence indicates that lncRNAs, like protein-coding genes, are transcribed mainly by RNA polymerase II; they are spliced products via canonical genomic splice site motifs, frequently ended with a poly A tail.
Here we report a novel PCR-based in vitro genomic DNA splicing strategy (designated as "PCR mediated Genomic DNA Splicing" or GDS strategy) for cloning of any eukaryotic cDNA or coding sequence from a genomic DNA preparation.
These successful cloning experiments suggest that our "Genomic DNA Splicing" technique is reliable and practicable.
The "Genomic DNA Splicing" protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours.
Our "Genomic DNA Splicing" technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns.
Here we reported a new gene synthesis strategy, "PCR mediated Genomic DNA Splicing" (GDS), which resembles "assembly PCR" in procedures but takes advantage of genomic DNA to avoid synthesis of large amount of oligonucleotides, thus is cost-effective and can reduce the unwanted mutations introduced by chemical synthesis of large numbers of relatively long oligonucleotides[10].
ACT-Graph nodes: the ACT-Graph nodes are created using one of the following—(i) Splices: genomic coordinates of splice start and end locations are obtained from spliced alignments; (ii) interpreting start and end sites of transcripts: we can use inference or annotations to identify these sites.
Many different PCR assays have targeted conserved and variable regions of kDNA minicircles [7], [8], [9], [10], [11], genomic DNA, splice leader mini-exon (SLME) [12], telomeric repeats [13], rRNA gene [14] and gp63 [15] for the detection of parasites directly from human tissues.
