Universal priming sequences
A 2500 base-pair long cDNA of unknown sequence was cloned into the MCS of a plasmid vector that has universal priming sequences adjacent to and on both sides of the MCS. On one side this is called the "forward universal priming sequence" and on the other it is called the "reverse universal priming sequence." Sanger sequencing was done using primers that are specific for each of these universal priming sequences and approximately 700 nucleotides of sequence was determined from each primer. Assuming it is not possible to obtain sequence analyses longer than 700 nucleotides, describe the steps that must be done to obtain the remainder of the sequence of the cloned cDNA.
Hint: There are two very different methods that will accomplish this. One method requires much more effort and is less reliable than the other. Either method will receive full credit.
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Easy way: Order two new sequencing oligos with sequences taken from near the last part (3' end) of the sequences already obtained with the two universal primers. If each of these is approximately 25 nt long, they are from within 50 nt of the end of the first sequences obtained, and they yield 700 nt of sequence each, then 625 of each result would be new sequence. This means that there would be at least 1400 nt (from the first set of sequencings) and 1250 nt more from the second set, or a total of 2650nt, enough to cover the entire 2500 bp long cDNA. Note: the exact lengths and spacings of the ...
Solution Summary
Universal priming sequences and sequencing cloned cDNA is provided in the solution.