Question
Experimental confirmation of semiconservative replication of $ \text{DNA}.$
✓
Answer
$(1)$ Matthew Meselson and Franklin Stahl $(1958)$ used equilibrium $–$ density $–$ gradient $–$ centrifugation technique to experimentally prove semiconservative $ \text{DNA}$ replication.
$(2)$ They cultured bacteria E.coli in the medium containing $^{14}N ($light nitrogen$).$ They obtained equilibrium density gradient band by using $6M \ CsCl_2.$ The position of this band is recorded.
$(3) \ E.$ coli cells were then transferred to $^{15}N$ medium $($heavy isotopic nitrogen$)$ and allowed to replicate for several generations. At equilibrium point density gradient band was obtained, by using $6M \ CsCl_2.$ The position of this band is recorded.
$(4)$ The heavy $ \text{DNA} (^{15}N)$ molecule can be distinguished from normal $ \text{DNA}$ by centrifugation in a $6M$ Cesium chloride $(CsCl_2)$ density gradient. At the equilibrium point $^{15}N \ \text{DNA}$ will form a band. In this both the strands of $ \text{DNA}$ are labelled with $^{15}N.$
$(5)$ Such $E.$ coli cells were then transferred to another medium containing $^{14}N$ i.e. normal $($light$)$ nitrogen. After first generation, the density gradient band for $^{14}N ^{15}N$ was obtained and its position was recorded. After second generation, two density gradient bands were obtained $–$ one at $^{14}N ^{15}N$ position and other at $^{14}N$ position.
$(6)$ The position of bands after two generations clearly proved that $ \text{DNA}$ replication is semiconservative.
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