I am reading an article about a novel splice-site mutation in the ELN gene that suggests an alternative mechanism for vascular elastinopathies.
Below, there is one part of the article which refers to methods used for determining the splicing mutation. I would like to know what exactly is cloning (mentioned in the second half of first paragraph) used for?
Here is the link to the whole article: A Novel Splice-Site Mutation in the ELN Gene Suggests an Alternative Mechanism for Vascular Elastinopathies
RNA Extraction, RT-PCR and Cloning To determine the effect of the splicing mutation, we per- formed RNA extraction using Trizol Reagent and the PureLink RNA Mini Kit (Thermo Scientific). We gener- ated cDNA using the SuperScript™ III First-Strand Synthesis System following the manufacturer’s recommendations. We carried out two PCR approaches to detect all pos- sible aberrant transcripts: a first one to generate a fragment containing exon 32 and the 3´UTR (primers F3 and R3), and a second one to generate a fragment spanning exon 30 and intron 31 (primers F1 and intR1) (Figures 3A and 4A). PCR primers were generated using Primer Blast (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/). The target regions of ELN were amplified by PCR and primer sequences are available upon request. For cloning, 1uL of PCR products were cloned into pCR™4-TOPO™ Vector (Invitrogen) using TOPO™ TA Cloning™, as the manufacturer’s instructions. Ligated plasmids were inserted in One Shot TOP10 E. coli (Invitrogen) through thermic shock during 30 sec. The bacteria were incubated during 1h in SOC medium. Transformed cells were plated on LB agar plates contain- ing 100 μg/mL ampicillin and were grown at 37°C over- night. Plasmid DNA was extracted from colonies with a Qiagen Plasmid Mini kit (Qiagen).
Sanger Confirmation We confirmed the mutation identified by WES and the cloned RT-PCR products using sanger sequencing. The genomic DNA encompassing the variant ELN-c.2132– 14_2161del was amplified by PCR using primers designed with the same tools mentioned previously (Primers avail- able upon request). The pCR™4-TOPO™ Vector plasmids containing the PCR products of RT-PCR were sequenced with T7 and T3 primers. The obtained sequences were compared to reference sequence ENST00000252034.12 (NM_000501.3). A detailed protocol is
available at: dx.doi.org/ 10.17504/protocols.io.bmcnk2ve