Q93 - Determining Insert Size and Orientation by Restriction Mapping

Theoretical B Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license

Recombinant DNA & Biotechnology Techniques

Mr. Trung cloned a coding sequence (CDS) of a gene into a vector and named the resulted plasmid as pVN2016. The CDS was inserted at the SacII recognition site which is located in the multi cloning site (MCS) region within the lacZ gene of the vector (Fig.Q.93A). The inserted CDS has a PstI restriction site located 0.8 kb upstream of its stop codon. To identify the size and direction of the inserted CDS, Mr. Trung digested this plasmid with different restriction enzymes, and the results of the digestions are shown in Fig.Q.93B.

Fig.Q.93 (A) A schematic map of the vector, showing the positions of the EcoRI, SpeI, and PstI recognition sites in the empty vector and the SacII cloning site. (B) A schematic electrophoresis gel of the digestion products from cutting pVN2016 with different single and combined restriction enzymes; M is a 1 kb DNA ladder.

Based on above data, indicate in the answer sheet if each of the following statements is true or false.

Figure 1. Figure 1.

Using the information and data, determine which of the statements are true or which are false.

A. The CDS is 2.6 kb in length and has an EcoRI recognition site at about 0.5 kb from one of its ends.

B. SpeI can be used to determine the orientation of the CDS.

C. The CDS is oriented in the same direction as lacZ.

D. If plasmid pVN2016 is digested by both enzymes SpeI and EcoRI in Tango buffer (EcoRI and SpeI cut at 100% and 20% efficiency, respectively), five fragments of 0.5, 0.8, 1.3, 2.1 and 3.0 kb could be detected by gel electrophoresis assuming that fragments smaller than 50 bp are not visible.

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Question reproduced from IBO 2016, Theoretical Paper B, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)