Biology, 15.04.2020 04:58 lailabirdiemae
You have a 1500 bp long cDNA in a plasmid vector. You want to sequence the cDNA insert. (You know the sequence of the vector.) The sequence reads you can obtain are only 1000 bp long.
How can you get the sequence of the entire cDNA insert?
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Synthesize sequencing primers that will hybridize adjacent to either end of the cDNA insert on different single-stranded circles. In two separate sequencing reactions, determine 1000 bp of sequence from each end of the insert. When turned into double strands virtually, the two sequences will overlap to form a 1500 bp contig. Synthesize sequencing primers that will hybridize adjacent to either end of the cDNA insert on different single-stranded circles. In two separate sequencing reactions, determine 1000 bp of sequence from each end of the insert. When turned into double strands virtually, the two sequences will overlap to form a 1500 bp contig.
Synthesize a sequencing primer that will hybridize adjacent to the insert and use it to obtain 1000 bp of sequence. Use that new sequence information to generate a second primer within the cDNA that will provide a read of the remainder of the 1500 bp sequence. Synthesize a sequencing primer that will hybridize adjacent to the insert and use it to obtain 1000 bp of sequence. Use that new sequence information to generate a second primer within the cDNA that will provide a read of the remainder of the 1500 bp sequence.
Synthesize two primers, one adjacent to each end of the insert. Add them both the same sequencing reaction in order to determine the sequence of the entire insert - 1000 bp from each side. Synthesize two primers, one adjacent to each end of the insert. Add them both the same sequencing reaction in order to determine the sequence of the entire insert - 1000 bp from each side.
Sequence 1000 bp from one end of the insert using a primer adjacent to one side. Compare that sequence to the RefSeq for the organism to find the remainder of the cDNA sequence. Sequence 1000 bp from one end of the insert using a primer adjacent to one side. Compare that sequence to the RefSeq for the organism to find the remainder of the cDNA sequence.
Make a mini-library of 200 bp overlapping fragments from the 1500 bp insert. Shotgun sequence library clones until you have a 1500 bp contig.
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