Cq

2'-deoxy ATP

5"

Ft CURE 20-13 Chain termination in the presence of dideoxynucleo tides.

In the top line is a DNA chain being extended at the 3' end with addition of an adenine nucleotide onto the previously incorporated cytostrie. The presence of dideoxycytosme in the growing chain (shown at the bottom) blocks furthei addition of incoming nucleotides as described in the text.

primer primer

5"

incorporate a 2'-,3'-dideoxynucleotide at the 3' end of a growing polynucleotide chain but once incorporated, the presence of the modified nucleotide causes elongation to terminate. The reason for this is the absence at the 3' end of the growing chain of a 3'-hydroxyl, which is needed for nucleophilic attack on the next incoming substrate molecule [Figure 20-13).

Now suppose that we "spike" a cocktail of the nucleotide substrates with the modified substrate 2'-,3'-dideoxyguanosine triphosphate (ddGTPJ at a ratio of one ddGTF molecule to 100 2'-deoxy-GTP molecules (dGTP). This will cause DNA synthesis to abort at a frequency of one in one hundred every time the DNA polymerase encounters a C on the template strand (Figure 20-14a). Because all of the DNA chains commence growth from the same point, the chain-terminating

"témplate strand" 1 2 3 Q 5 e Q 8 9 10 Q 12 _A 1 G C G A C T T A C 5'

primer

nucleotide length

DMA synthesis substrates: dQB dSE ci EH GE

ddS5)

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