Cloning Polymerase Chain Reaction (PCR) Products: TOPO TA Cloning

This protocol describes the use of TOPO-activated TA vectors for cloning. Manufacturers of cloning kits provide excellent manuals that explain in detail what to do and why to do it. This makes TOPO cloning easy, but not foolproof. When setting up TOPO cloning for the first time, set up a trial experiment as described here.

Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning

Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient’s peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.

Coexistence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in Pseudomonas aeruginosa Serogroup O2 and Their Contributions to Cell Surface Diversity

ABSTRACT Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy α product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are α-glycosidically linked, while those in O2 and O16 are β-linked. We hypothesized that a derivative of the D3 bacteriophage wzy β is present in the chromosomes of O2 and O16 and that this gene is responsible for the β-linkage. By a combination of PCR and primer walking, wzy β genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy β was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy β is capable of complementing the O16 wzy β mutant, as well as cross-complementing a wzy α knockout mutant. However, in the latter case, the restored O antigen was β-linked. Using reverse transcription-PCR, we showed that wzy α was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy α mutant of O5. With the coexistence of wzy α and wzy β in O2 and O16 and the B-band O polysaccharides in these being β-linked, we hypothesized that iap, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of iap in both O2 and O16 serotypes.