PCR and Dideoxy Sequencing Video Lessons

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Problem: In order to determine if a mutation has occured in Gene X, DNA is sequenced using the Sanger method. Briefly explain how this method works and then give the sequence of DNA (10 bases) that is represented by the following gel. Be sure to label the 5' and 3' end of the DNA.Lane 1: dATP, dGTP, dCTP, dTTP, ddATPLane 2: dATP, dGTP, dCTP, dTTP, ddGTPLane 3: dATP, dGTP, dCTP, dTTP, ddTTPLane 4: dATP, dGTP, dCTP, dTTP, ddCTP

FREE Expert Solution

Sanger method is a sequencing method that employs chain termination. This utilizes the chain elongation of the DNA polymerase but causes premature termination in specific bases per well. This premature termination is due to the presence of dideoxynucleoside triphosphates (ddNTPs). They can serve as the bases for chain elongation but once they attach, their lack of 3' OH group makes it impossible for elongation to continue. This causes the formation of differently sized fragments, thus enables the determination of the sequence. 

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Problem Details

In order to determine if a mutation has occured in Gene X, DNA is sequenced using the Sanger method. Briefly explain how this method works and then give the sequence of DNA (10 bases) that is represented by the following gel. Be sure to label the 5' and 3' end of the DNA.

Lane 1: dATP, dGTP, dCTP, dTTP, ddATP

Lane 2: dATP, dGTP, dCTP, dTTP, ddGTP

Lane 3: dATP, dGTP, dCTP, dTTP, ddTTP

Lane 4: dATP, dGTP, dCTP, dTTP, ddCTP

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