Question

6.- Your friend Silvester wishes to subclone a 1.7 kb DNA fragment into the pCR 2.1 TOPO plasmid vector. His goal is to amplify the desired fragment by PCR. At the end of the PCR cycling reaction, he immediately adds 4 ?L of PCR reaction, (the maximum of DNA allowable) to the ligation mix. He did the transformation and plated it onto solid media containing the right amount of X-gal and ampicillin.

He picked a single white colony, inoculated it into liquid LB media with ampicillin and grew it up overnight. The next day he purified the plasmid DNA from the colony and sent it for sequencing. He prepared two tubes of DNA, one sample of plasmid will be sequenced using the M13 -20 primer to sequence the bottom strand. The second DNA sample will be sequenced with the M13 Reverse primer to sequence the upper strand. Silvester got the sequence back and the chromatograms were very good. When he BLAST’ed the sequences using NCBI he realized that he had cloned a PCR product that was only 800 bp. He was upset because he felt that since he'd used blue-white selection and chose a white colony that he should have isolated the fragment he was interested in. He is confused and does not understand his results.

(i) Why was Silvester able to conclude with confidence that the PCR product he submitted for sequencing was only 800 bp? (10 pts).

            (ii) Explain why a 800 bp sequence was ligated into the pCR 2.1 TOPO vector although his aim was to amplify a larger PCR product (5 pts).

            (iii) Give him some recommendations that would help to him to select the right clones for sequencing in the future (15 pts).

Answer 1

(i) Ans- NCBI Basic Local Alignment Search Tool (BLAST) recognize the similarity between sequence within the local region. When there is an exact homology between two sequences then the BLAST gives good hits for the query sequence. In the above-mentioned case, Silvester runs BLAST with his PCR product (sequence). While analyzing the BLAST result he probably gets a good hit on a chromosome but the hit length always remain fixed i.e. 800bp. So, Silvester concludes that the length of his PCR product should be 800bp.
(ii) Ans- 800 bp ligation might occur due to the improper restriction digestion and the ratio of the insert and the vector might also be responsible for the inappropriate ligation

fig: Cloning of PCR product by TA overhangs

(iii)-Ans- The ratio of the vector and insert is very crucial for a proper ligation. For sticky end ligation generally 3:1 (insert: vector) ration gives a good result. in case of blunt end ligation the increased insert ratio is recommended. Choosing of the restriction enzyme is also very crucial for a directional cloning.