Understand the experiments of Erwin Chargaff and what he discovered. Learn Erwin Chargaff's Rules about DNA's nitrogenous bases. Find what he contributed to DNA.
Updated: 11/21/2023
Erwin Chargaff's Experiment | Overview, Discoveries & Rules
Table of Contents
- The Experiments of Erwin Chargaff
- What Did Chargaff Discover About DNA?
- Erwin Chargaff's Rules
- Lesson Summary
Frequently Asked Questions
What did Erwin Chargaff discover and why was this important?
Chargaff carried out a series of experiments which provided two important insights regarding the four nitrogenous bases. First of all, the proportion of the four bases varies from species to species. Second, the ratios of A:T and C:G are always 1:1.
How did Chargaff determine the base pairing in DNA?
Chargaff laid the foundation for determining base pairing in DNA by observing the amount of the four nitrogenous bases found in different samples of DNA. Paper chromatography was used to separate the substances found in DNA and UV spectrophotometry was used to count the amount of each base found in each sample.
What are Erwin Chargaff's two rules?
Erwin Chargaff developed two rules based on his research. First of all, the proportion of the four nitrogenous bases varies from species to species. Second, the ratios of A:T and C:G are always 1:1.
Table of Contents
- The Experiments of Erwin Chargaff
- What Did Chargaff Discover About DNA?
- Erwin Chargaff's Rules
- Lesson Summary
Erwin Chargaff was an Austrian-Hungarian biochemist born in Czernowitz, Austria who developed the Chargaff Rules. These rules helped to determine and established the pattern of nitrogenous base pairing in DNA.
From an early age, Chargaff possessed a keen interest in chemistry. After graduating from the University of Vienna, he decided to pursue a doctoral program in chemistry. Upon the conferral of his doctoral degree in 1928, Chargaff moved to the United States in order to find a suitable research position. He eventually became a professor of biochemistry at the medical school at Columbia University.
In the 1940s, three biochemists, Oswald Avery, Colin MacLeod, and Maclyn McCarty, performed a series of experiments demonstrating that DNA represented the genetic material responsible for transforming non-virulent bacteria into virulent bacteria. As a result, Avery, MacLeod, and McCarty determined that genes were made of DNA and that DNA represented the genetic code used in the transmission of traits. Upon reading the results of their research, Chargaff developed a keen interest in determining the ''language'' involved in the construction of DNA and the transmission of heritable traits.
In order to elucidate this ''language,'' Chargaff developed an experiment in order to identify the different units responsible for gene coding in DNA. Scientists of this period already contended that DNA was formed by equal amounts of the four nitrogenous bases- adenine, cytosine, guanine, and thymine. This was referred to as the tetranucleotide hypothesis. Scientists at this time also believed as part of the tetranucleotide hypothesis that the proportion of bases in DNA was the same in all species. Chargaff wanted to determine whether the tetranucleotide hypothesis was true by looking for the amount of each nitrogenous base in samples of DNA taken from different species.
At that time, DNA was difficult to acquire, so the Chargaff experiment needed to find a way for determining the amount and type of material found in small quantities of DNA. Using a recently developed procedure involving paper chromatography, Chargaff used the photoelectric properties of the particles in DNA in order to identify the quantities of each base.
First of all, Chargaff used paper chromatography to separate DNA into its constituent particles. Paper chromatography works by examining the rates at which different substances travel when exposed to a solvent. Differences in the distance travelled by each substance can be used to determine the identities for each component in a mixture. Next, Chargaff used mercuric nitrate in order to fix each of these separated particles as mercury salts. Finally, Chargaff used the amount of ultraviolet absorption to determine the amount of each nitrogenous base present in a sample.
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What did Chargaff discover about DNA as a result of his experiment? In the following sections, Erwin Chargaff's discoveries will be further discussed.
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Chargaff's First Discovery: Species and Bases
Chargaff's first major discovery as a result of these experiments showed that different species of organisms possessed different amounts of the four nitrogenous bases. This challenged the assumptions associated with the tetranucleotide hypothesis. Moreover, although the genetic code was only made from four bases, Chargaff concluded that there were numerous ways to arrange these bases, thus allowing for the diversity of genetic traits found in different species.
Chargaff's Second Discovery: Fixed Ratios and Bases
Chargaff's second discovery involved the ratios of the four nitrogenous bases. Although DNA samples were taken from different species, the experiments showed that there was a fixed ratio of bases in each sample. In other words, there was always an equal amount of adenine and thymine, as well as cytosine and guanine, in each sample of DNA. Moreover, Chargaff stated that despite differences in the amounts of each base found within any given species. the ratio of A:T and C:G was always 1:1 in all species.
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Although this hinted at the existence of complementary base pairing between adenine:thymine and cytosine:guanine, Chargaff never explicitly discussed this concept in his published research. However, subsequent scientists, such as Watson and Crick, built upon Chargaff's research in order to develop these rules for base pairing.
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Paper Chromatographs
As mentioned earlier, paper chromatography uses the distance traveled by different substances when exposed to a solvent in order to determine the identify of each component. When using paper chromatography, a sample from a compound is placed upon one end of a sheet of filter paper. Then the opposite end of the filter paper is placed in a solvent. As the solvent breaks down the compound, the different components will travel up the filter paper at different rates. Using this method, Chargaff separated the different components found in DNA and discovered that adenine, guanine, thymine, and cytosine existed in fixed ratios and that each species possessed a different proportion of each base pair.
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UV Spectrophotometry
UV spectrophotometry relies upon differences in the amount of light absorbed by different compounds. This method specifically measures the intensity of light that is absorbed or reflected by different substances. Chargaff used UV spectrophotometry in order to observe the amount of light absorbed by the separated compounds found on each strip of filter paper. In order to control for differences in UV absorption, Chargaff exposed each sample to different wavelengths of UV light. Then he calculated the amount of each nitrogenous base using the differences between the maximum absorption and a baseline wavelength values for each base.
The results of UV spectrophotometry showed that adenine and thymine occurred in equal amounts. Guanine and cytosine were also found to occur in equal amounts.
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Chargaff repeated this experiment using samples taken from different species. Based upon the results of these experiments, Erwin Chargaff's contribution to the scientific understanding of DNA is known as the Chargaff Rules. These rules state that:
- The amount of adenine is always equal to the amount of thymine found in a sample
- The amount of cytosine is always equal to the amount of guanine found in a sample.
- The proportion of the four nitrogenous bases varies from species to species.
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Erwin Chargaff is an early 20th century biochemist born in the former Austro-Hungarian Empire. He graduated from the University of Vienna, pursued a doctoral degree in chemistry, and became a professor of biochemistry at Columbia University, where his groundbreaking work on DNA was conducted. Through a series of experiments conducted in the 1940s, Chargaff tested the prevailing early 20th century hypothesis that the four nitrogenous bases were found in the same proportion in all species.
Chargaff's experiment relied upon the use of paper chromatography and UV spectrophotometry. Chargaff used paper chromatography to separate out the different chemical substances found in samples of DNA. Then, UV spectrophotometry was used to calculate the exact amounts of adenine, thymine, cytosine, and guanine found in DNA samples. Based upon the results from these experiments, Chargaff discovered that different species have different relative amounts of the four nitrogenous bases and that the ratios of A:T and C:G are always 1:1.
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Video Transcript
Who Was Erwin Chargaff?
Many of the facts about DNA that we now consider to be common knowledge, such as its double helix structure and its role in coding genetic information, came about because of an Austro-Hungarian biochemist named Erwin Chargaff. Chargaff was born in Austria in 1905 and fleeing Jewish persecution throughout Europe, he worked in Vienna, Connecticut, Berlin, and Paris before settling in New York at Columbia University in 1935. He permanently relocated to the United States, becoming a citizen in 1940 and dying in New York in 2002. It was at Columbia where his groundbreaking research on the structure of DNA took place.
Chargaff's Experiments
In 1944, Chargaff read a paper by Oswald Avery proposing the idea that DNA coded and transmitted genetic information. Though many scientists disagreed with Avery's conclusions, Chargaff was inspired. He dropped all of his previous research to focus on studying DNA full-time.
Paper Chromatography
That same year, a new technique called paper chromatography was developed. Using paper chromatography, chemists can separate out the different chemical substances that are found within a solution.
Let's take a look at how this is done. First, a sample of the solution in question is dropped onto the edge of a sheet of filter paper. The filter paper is then lowered into a liquid called a solvent, which is slowly absorbed up through the paper in the same manner that water is absorbed by a paper towel.
The solvent is able to dissolve the different components of the sample solution. As it passes through the solution, the solvent picks up the various substances and takes them along for the ride.
Some of these substances will be more attracted to the solvent than the paper, so they move up the filter paper quickly. Other substances will be more attracted to the paper, so these move up the filter paper more slowly. As long as all of the substances you are interested in have a different level of attraction to either the solvent or the paper, they will be pulled up the paper at different rates and will thus become separated from each other.
Chargaff adapted the paper chromatography technique to separate out the four nitrogenous bases of DNA. The four bases of DNA - adenine, guanine, thymine and cytosine - are the parts of the molecule that do the actual coding of our genes. Now that he had each of the bases isolated, he used another budding technology, UV spectrophotometry, to calculate their relative amounts.
UV Spectrophotometry
All matter absorbs and reflects light. Our vision takes advantage of this: when we perceive objects as having certain colors, what we are actually seeing is certain wavelengths of visible light being absorbed while other wavelengths are reflected back. For example, the chlorophyll in plant leaves absorbs all the colors of the spectrum except green. The green wavelengths are reflected back to our eyes, and we perceive the plants as green.
Similarly, the DNA bases that we discussed earlier absorb light in the ultraviolet range. Since Chargaff was interested in observing and measuring the DNA bases, he utilized the technique of UV spectrophotometry.
In UV spectrophotometry, a particular wavelength of UV light is isolated and shined at a solution containing an unknown quantity of chemical compounds, and the amount of light absorbed by the solution is compared to the amount of light that passes through. If a large percentage of the light is absorbed, this indicates a high concentration of the compound you're observing. If a small percentage of the light is absorbed, this signifies a low concentration of the compound. You can then use this data to calculate the exact number of molecules in your sample.
So, Chargaff first isolated the four different nitrogenous bases of DNA referred to as A, G, T, and C, using paper chromatography. He then created new solutions containing each of his isolated samples and observed them using UV spectrophotometry. With the data he obtained from the UV spectrophotometer, he calculated the exact amount of each base found in his samples.
Chargaff's Rules
Chargaff repeated these experiments using the DNA of many different organisms, including people, plants, fish, bacteria, and fungi. He made several radical discoveries, which he first published in 1950. The first was that different species had different ratios of each of the bases. His results for humans, rats, and maize are shown in this table:
| A | G | T | C | A:T | G:C | |
|---|---|---|---|---|---|---|
| Human | 29.3% | 20.7% | 30.0% | 20.0% | 0.98 | 1.04 |
| Rat | 28.6% | 21.4% | 28.4% | 20.5% | 1.01 | 1.00 |
| Maize | 26.8% | 22.8% | 27.2% | 23.2% | 0.99 | 0.98 |
The second discovery can also be observed in the table. Chargaff noticed that, regardless of the species, the amount of adenine was always nearly identical to the amount of thymine, and the amount of guanine was always nearly identical to the amount of cytosine.
Chargaff's discoveries are summarized in what we now refer to as Chargaff's rules:
- In any species, the ratio of A:T is 1:1 and the ratio of G:C is 1:1
- The relative amounts of A, G, T, and C vary from one species to another
Lesson Summary
To recap, Erwin Chargaff's discoveries, what we now call Chargaff's rules, were essential building blocks to our current understanding of DNA. Thanks to his early experiments using paper chromatography and UV spectrophotometry, Chargaff's findings directly led to the future discoveries of the structure of the DNA molecule, as well as how DNA replicates and codes genetic information.
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