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The Meselson-Stahl Experiment: Demonstrating Semiconservative DNA Replication, Schemes and Mind Maps of Biochemistry

The meselson-stahl experiment, a groundbreaking study in biochemistry that provided evidence for the semiconservative replication of dna. The experiment involved growing e. Coli bacteria in media with heavy nitrogen isotope 15n and then transferring them to media with the light nitrogen isotope 14n. By analyzing the dna using cscl density gradient centrifugation, meselson and stahl observed hybrid dna molecules containing one old and one new strand after one generation and two distinct bands of light and hybrid dna after two generations, supporting the semiconservative model of dna replication.

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Biochemistry
I N PE R S PE C T IV E
The MeselsonStahl Experiment
DNA replication occurs before every cell division. The mechanism by which DNA
copies are synthesized is similar in all living organisms. After the two strands have
separated, each serves as a template for the synthesis of a complementary strand.
(In other words, each of the two new DNA molecules contains one old strand and
one new strand.) This process, referred to as semiconservative replication, was first
demonstrated in an elegant experiment reported in 1958 by Matthew Meselson and
Franklin Stahl (Figure 1).
In this classic work, Meselson and Stahl took advantage of the increase in
density of DNA labeled with the heavy nitrogen isotope 15N (the most abundant
nitrogen isotope is 14N). After E. coli cells were grown for 14 generations in growth
media whose nitrogen source consisted only of 15NH4Cl, the 15N-containing cells
were transferred to growth media containing the 14N isotope. At the end of both one
and two cell divisions, samples were removed. The DNA in each of these samples
was isolated and analyzed by CsCl density gradient centrifugation. (Refer to
Biochemical Methods 2.1 for a description of density gradient centrifugation.)
Because pure 15N-DNA and 14N-DNA produce characteristic bands in centrifuged
CsCl tubes, this analytical method discriminates between DNA molecules containing
large amounts of the two nitrogen isotopes. When the DNA isolated from 15N-
containing cells grown in 14N medium for precisely one generation was centrifuged,
only one band was observed. Because this band occurred halfway between where
15N-DNA and 14N-DNA bands would normally appear, it seemed reasonable to
assume that the new DNA was a hybrid molecule, that is, it contained one 15N strand
and one 14N strand. (Any other means of replication would create more than one
band.) After two cell divisions, extracted DNA was resolved into two discrete bands
of equal intensity, one made up of 14N,14N-DNA (light DNA) and one made up of
hybrid molecules (14N,15N-DNA), a result that also supported the semiconservative
model of DNA synthesis.
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Biochemistry I N P E R S P E C T I V E

The Meselson–Stahl Experiment

DNA replication occurs before every cell division. The mechanism by which DNA copies are synthesized is similar in all living organisms. After the two strands have separated, each serves as a template for the synthesis of a complementary strand. (In other words, each of the two new DNA molecules contains one old strand and one new strand.) This process, referred to as semiconservative replication, was first demonstrated in an elegant experiment reported in 1958 by Matthew Meselson and Franklin Stahl ( Figure 1 ). In this classic work, Meselson and Stahl took advantage of the increase in density of DNA labeled with the heavy nitrogen isotope 15 N (the most abundant nitrogen isotope is 14 N). After E. coli cells were grown for 14 generations in growth media whose nitrogen source consisted only of 15 NH 4 Cl, the 15 N-containing cells were transferred to growth media containing the 14 N isotope. At the end of both one and two cell divisions, samples were removed. The DNA in each of these samples was isolated and analyzed by CsCl density gradient centrifugation. (Refer to Biochemical Methods 2.1 for a description of density gradient centrifugation.) Because pure 15 N-DNA and 14 N-DNA produce characteristic bands in centrifuged CsCl tubes, this analytical method discriminates between DNA molecules containing large amounts of the two nitrogen isotopes. When the DNA isolated from 15 N- containing cells grown in 14 N medium for precisely one generation was centrifuged, only one band was observed. Because this band occurred halfway between where (^15) N-DNA and 14 N-DNA bands would normally appear, it seemed reasonable to

assume that the new DNA was a hybrid molecule, that is, it contained one 15 N strand and one 14 N strand. (Any other means of replication would create more than one band.) After two cell divisions, extracted DNA was resolved into two discrete bands of equal intensity, one made up of 14 N,^14 N-DNA (light DNA) and one made up of hybrid molecules (^14 N,^15 N-DNA), a result that also supported the semiconservative model of DNA synthesis.

FIGURE 1

The Meselson–Stahl Experiment CsCl centrifugation of E. coli DNA can distinguish heavy DNA grown in 15 N media (1) from light DNA grown in 14 N media (2). A mixture is shown in (3). When E. coli cells enriched in (^15) N are grown in 14 N for one generation, all genomic DNA is of intermediate density (4). After two generations, half of the DNA is light and half is of intermediate density (5). After three generations, 75% of the DNA is light and 25% of the DNA is of intermediate density (6).