Iiy

no 3'-OH group to form a phosphodiester bond with an incoming nucleotide. Thus, ddNTPs terminate DNA synthesis.

A single DNA molecule cannot be sequenced; so any DNA fragment to be sequenced must first be amplified by PCR or by cloning in bacteria. Copies of the target DNA are isolated and split into four parts (IFigure 19.6). Each part is placed in a different tube, to which are added:

1. many copies of a primer that is complementary to one end of the target DNA strand;

2. all four deoxyribonucleoside triphosphates (dCTP, dATP, dGTP, and dTTP), the normal precursors of DNA synthesis;

3. a small amount of one of the four types of dideoxyribonucleoside triphosphates (ddCTP, ddATP, ddGTP, or ddTTP), which will terminate DNA synthesis as soon as it is incorporated into any growing chain (each of the four tubes received a different ddNTP); and

4. DNA polymerase.

Either the primer or one of the dNTPs is radioactively or chemically labeled so that newly produced DNA can be detected.

Within each of the four tubes, the DNA polymerase enzyme carries out DNA synthesis. Let's consider the reaction in one of the four tubes; the one that received ddATP. Within this tube, each of the single strands of target DNA

ij Each of four reactions <

; contains single-stranded target DNA to be sequenced,...

Template Primer

; contains single-stranded target DNA to be sequenced,...

+ ddCTP

^ When a dideoxynucleotide is incorporated into the growing chain, synthesis terminates because the dideoxynucleotide lacks a 3' OH.

Template â– CTAAGCTCGACTl ddA

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