Lec 15 DNA Sequencing

8/9/2019 Lec 15 DNA Sequencing

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DNA Sequencing

How do you do it?


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DNA Sequencing

DNA sequencing used to determine theactual DNA sequence of an organism.

Using a computer, one can identify an organism,and predict protein sequences and functions

based on the nucleic acid data.The most commonly used sequencing method isthe dideoxy method.

This method uses dideoxynucleotide triphosphates(ddNTPs)which have an H on the 3 carbon of the ribose sugar insteadof the normal OH found in deoxynucleotide triphosphates(dNTPs).

Dideoxynucleotides are chain terminators.

In a synthesis reaction, if a dideoxynucleotide is added insteadof the normal deoxynucleotide, the synthesis stops at that

point because the 3OH necessary for the addition of the nextnucleotide is absent.


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Deoxy versus dideoxy


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DNA synthesis


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DNA sequencing continued

In the dideoxy method of sequencing, the template DNA thatis to be sequenced is mixed with a primer complementary tothe template DNA and the four normal dNTPs, one of whichis radioactively labeled for subsequent visualizationpurposes.

This mixture is then splint into four different tubes that arelabeled A, C, G, and T. Each tube is then spiked with adifferent ddNTP (ddATP for tube A, ddCTP for tube C,ddGTT for tube G, or ddTTP for tube T).

DNA polymerase is added and using the DNA template and

its complementary primer, the synthesis of new strands ofDNA complementary to the template begins.

Occasionally a dideoxynucleotide is added instead of thenormal deoxynucleotide and synthesis of that strand isterminated at that point.


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In the tube containing ddATP, some percentage of newlysynthesized molecules will get a ddATP in each place thatthere is a T in the template DNA.

The result is a set of new DNA molecules in tube A, each ofwhich ends in an A.

A similar type of reaction occurs in the three other tubes toresult in molecules that end in C, G, and T in tubes C, G,and T respectively.

After the synthesis reactions are complete, the products ofthe four different tubes are loaded onto four adjacent lane of

a polyacrylamide gel and the different fragments areseparated by size.

The sequencing gel is able to resolve fragments that differ insize from each other by only one base.


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After electrophoresis to separate the fragments bysize, the fragments are visualized to exposing thegel to photographic film (Remember that onenucleotide was radioactively labeled).

All fragments in lane A will end in an A, fragmentsin lane C will all end in a C, fragments in lane Gwill all end in a G, and fragments in lane T will allend in a T.

The sequence of the DNA is read from the gel bystarting at the bottom and reading upward.


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Dideoxy DNA Sequencing


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DNA sequencing


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DNA sequencing

Automated DNA sequencing in automated DNAsequencing a radioactive deoxynucleotide is not usedand all four dideoxy reactions are done in a singletube.

This is possible because each ddNTPs is labeled witha different flourescent dye.

Therefore the dye present in each synthesizedfragment corresponds to the dye attached to the

dideoxynucleotide that was added to terminate thesynthesis of that particular fragment.

The contents of the single tube reaction are loadedonto a single lane of a gel and electrophoresis isdone.


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DNA Sequencing

A flourimeter and computer are hooked up tothe gel and they detect and record the dyeattached to the fragments as they come off

the gel.The sequence is determined by the order ofthe dyes coming off the gel.


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Automated DNA sequencing

Lec 15 DNA Sequencing

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