Sanger DNA Sequencing Exam Questions and Answers Already Passed
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Course
Sanger DNA Sequencing
Institution
Sanger DNA Sequencing
Sanger DNA Sequencing Exam Questions and Answers Already Passed
DNA Sequencing - Answers the primary method for gne and protein characterization
Sanger Sequencing AKA - Answers - Dideoxynucleotide sequencing
- Chain termination sequencing (DNA synthesis will terminate with the incorporation o...
- Chain termination sequencing (DNA synthesis will terminate with the incorporation of a
dideoxynucleotide b/c no more 5'-3' synthesis possible)
Principle of Sanger Sequencing - Answers - All four dNTPs are used
- Four separate reactions are set up
- Each as a different ddNTP: ddATP, ddTTP, ddCTP, ddGTP
- Klenow will incorporate dNTPs until a ddNTP is incorporated which terminates synthesis
- DNA is denatured and the four smaples are loaded next to each other and DNA separted on a PAGE gel
- Separate based on size of fragment -> run on a high % page gel to separate very specifically (even
single base differences)
Steps of sanger sequencing - Answers 1. PCR clean up
(remove excess primer by exonucleas 1 and dNTP's by alkaine prophasphatase
2. Cycle sequencing
3. Purify the extension products: ethanol precipitation '
4. sequencing analaysis '
5. gel or capillaries
6. Analysis
A-T cloning - Answers -In a PCR-reaction most polymerases add A to the end of the product
-Vector has a T-overhang where the PCR-poduct is ligated.
-Following ligation, the vector with the PCR-product is transformed into competent E.coli cells by heat-
shock
-The E.coli is plated on to selective agar plates
-Pick colonies (clones) and culture
-Isolate plasmid-DNA
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