1.1 (12 pts) You are assigned to design a biosensor for multi-target detection against dengue virus type 1 and type 2, and African swine fever virus by detecting the specific sequences of their RNAs or DNAs. Design three different DNA probes to recognize the 25-nt long nucleic acids of specific sequences highlighted below. Label the DNA terminals and start from 5’ terminal. Dengue virus type 1: 10,550–10,574 (M87512) 5’… 10501 agaggagacc cctcccaaaa cataacgcag cagcggggcc caacaccagg ggaagctgta 10561 tcctggtggt aaggactaga ggttagagga gacccccggc ataacaataa acagcatatt 10621 gacgctggga gagaccagag atcctgctgt ctctacagca tcattccagg cacagaacgc … 3’ Dengue virus type 2: 10,557–10,581 (M20558) 5’… 10501 ggttagagga gacccctccc ttacaaatcg cagcaacaac gggggcccaa ggtgagatga 10561 agctgtagtc tcactggaag gactagaggt tagaggagac ccccccaaaa caaaaaacag 10621 catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca … 3’ African swine fever virus E75 (FN557520) 5’ … 99061 aggtaagctt gtttcccaag gtgggggtac ccgtatgcgg gcgtacttta ttgtattcaa 99121 accctactgg aacataaggc ttaaaatgcg cattaaaatg aaccaaatgt gtttcttcga 99181 tttgactcaa agtgggttcg gggtcgggtt tcccataact tttgttcaca tttttaatgt … 3’ (www.ncbi.nlm.nih.gov/genbank/collab) GenBank format by historical convention displays RNA sequences using the DNA alphabet. Thus, for the convenience of people reading GenBank records, the genetic code tables shown here use T instead of U.
1.2 (5 pts) Now you have the sensors functionalized with DNA probes and are ready for detecting against the viral RNA. In practice, hybridization of target viral RNA to the DNA probes is typically carried out at 37 °C for 20 min in a hybridization buffer solution which contains 750 mM NaCl, and 75 mM sodium citrate, a high ionic strength (very salty) buffer solution. Why such a high ionic strength buffer is preferred for most of the hybridization process? Explain it using the ion screening effect and intermolecular force we discussed in the class.
1.3 (a) (8 pts) Use the Tm calculator on this website (http://www.biophp.org/minitools/melting_temperature/demo.php) to find the melting temperatures of these three hybridized DNA-RNA fragments (25 bps) under 50 mM salt concentration. (Keep primer concentration = 200 nM and Mg2+ = 0 mM)
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