Describ tbe experiment that help demonstrate semi conservative mode of dna replication
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Heya..!
Semiconservative replication of DNA was proved by the work of Mathew Meselson and Franklin stahl in 1958.
They grew Escherichia coli for many generations in a medium having heavy isotope of nitrogen in the form of 15N , till the bacterial DNA became completely labelled with heavy isotope.
The labelled bacteria were then shifted to fresh medium having normal or 14N nitrogen.
Samples were taken for each generation (one generation takes 20 minutes as E. coli divides inin 20 minutes) and the DNA was tested for heavy isotope of nitrogen through DENSITY GRADIENT CENTRIFUGATION using caesium chloride.
Caesium chloride is a highly water soluble heavy salt. When spun in centrifuge at high speed (almost 50,000 revolutions per minute) the salt forms a density gradient with heaviest most concentrated region at the bottom and successively less concentrated lighter one towards the surface.
When DNA is mixed with caesium chloride it will settle down at particular height in centrifugation, heavier towards the base and the lighter one higher up.
A fluorochrome called ethidium bromide is used to enhance contrast as flourochrome is specific for DNA.
Meselson and Stahl found that DNA of the first generation was hybruid or intermediate type. (one strand made entirely by 15N and the otherone by 14N.
It settled in caesium chloride solution ata level higher than the fully labelled DNA of parent bacteria( both the strands made of 15N).
The second generation of bacteria after next 20 minutes, containes two types of DNA 50% light(14N and 14N) AND 50% Intermediate type(14N AND 15N).
the third generation of bacteria after next 20 minutes contained two types of DNA 25% intermediate (14N &15N) and rest 75% was normal type (14N &14N) in 1:3 ratio.
The fourth generation after next 20 minutes contained 12.5% of intermediate type and rest 87.5% as normal type in 1:7 ratio.
------CONCLUSION--------
This observation is possible only if two strands of DNA duplex seperate at the time of replication and act as a template for the synthesis of new complemantary strands of DNA having normal or 14N. This will produce two DNA duplexes with one old strand (15N) and one new strand (14N). During the formation of second generation, 15N and 14N strands seperate to function as templates so that 50% of new DNA duplexes posses only normal or 14N strands while another 50% have both 15N& 14N. In this way at each replication, one strand of parent DNA is conserved in the daughter while the second is freshly synthesised.
HOPE THIS HELPS YOU.
REFER TO THE IMAGE ATTACHED.
BE BRAINLY
Semiconservative replication of DNA was proved by the work of Mathew Meselson and Franklin stahl in 1958.
They grew Escherichia coli for many generations in a medium having heavy isotope of nitrogen in the form of 15N , till the bacterial DNA became completely labelled with heavy isotope.
The labelled bacteria were then shifted to fresh medium having normal or 14N nitrogen.
Samples were taken for each generation (one generation takes 20 minutes as E. coli divides inin 20 minutes) and the DNA was tested for heavy isotope of nitrogen through DENSITY GRADIENT CENTRIFUGATION using caesium chloride.
Caesium chloride is a highly water soluble heavy salt. When spun in centrifuge at high speed (almost 50,000 revolutions per minute) the salt forms a density gradient with heaviest most concentrated region at the bottom and successively less concentrated lighter one towards the surface.
When DNA is mixed with caesium chloride it will settle down at particular height in centrifugation, heavier towards the base and the lighter one higher up.
A fluorochrome called ethidium bromide is used to enhance contrast as flourochrome is specific for DNA.
Meselson and Stahl found that DNA of the first generation was hybruid or intermediate type. (one strand made entirely by 15N and the otherone by 14N.
It settled in caesium chloride solution ata level higher than the fully labelled DNA of parent bacteria( both the strands made of 15N).
The second generation of bacteria after next 20 minutes, containes two types of DNA 50% light(14N and 14N) AND 50% Intermediate type(14N AND 15N).
the third generation of bacteria after next 20 minutes contained two types of DNA 25% intermediate (14N &15N) and rest 75% was normal type (14N &14N) in 1:3 ratio.
The fourth generation after next 20 minutes contained 12.5% of intermediate type and rest 87.5% as normal type in 1:7 ratio.
------CONCLUSION--------
This observation is possible only if two strands of DNA duplex seperate at the time of replication and act as a template for the synthesis of new complemantary strands of DNA having normal or 14N. This will produce two DNA duplexes with one old strand (15N) and one new strand (14N). During the formation of second generation, 15N and 14N strands seperate to function as templates so that 50% of new DNA duplexes posses only normal or 14N strands while another 50% have both 15N& 14N. In this way at each replication, one strand of parent DNA is conserved in the daughter while the second is freshly synthesised.
HOPE THIS HELPS YOU.
REFER TO THE IMAGE ATTACHED.
BE BRAINLY
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