Structure of DNA
Inquire: DNA Structure and Sequencing
The currently accepted model of the double-helix structure of DNA was proposed by James Watson and Francis Crick. Some of the most recognizable features are that the two strands that make up the double helix have complementary base sequences and anti-parallel orientations. Essentially, a strand of DNA looks like a ladder that has been twisted. Alternating deoxyribose sugars and phosphates form the backbone (or the sides of the ladder), and the nitrogenous bases are stacked like rungs inside. The nitrogenous bases are paired together in a pattern. A purine always pairs with a pyrimidine; A pairs with T, and G pairs with C. One turn of the helix has 10 base pairs. The DNA structure of prokaryotes are much simpler than eukaryotes in many of their features. Most prokaryotes contain a single, circular chromosome. In general, eukaryotic chromosomes contain a linear DNA molecule packaged into nucleosomes, and have two distinct regions that can be distinguished by staining, reflecting different states of packaging and compaction.
What is the molecular structure of DNA?
Watch: The DNA Race
Read: DNA Structure and Sequencing
The currently accepted model of the structure of DNA was proposed in 1953 by James Watson and Francis Crick, who made their model after seeing a photograph of DNA that Rosalind Franklin had taken using X-ray crystallography. The photo showed the molecule’s double-helix shape and dimensions. The two strands that make up the double helix are complementary and anti-parallel in nature. That is, one strand runs in the 5′ to 3′ direction, whereas the complementary strand runs in the 3′ to 5′ direction. (The significance of directionality will be important when we explore how DNA copies itself.) DNA is a polymer of nucleotides that consists of deoxyribose sugar, a phosphate group, and one of four nitrogenous bases — A, T, C, and G — with a purine always pairing with a pyrimidine (as Erwin Chargaff found). The genetic “language” of DNA is found in sequences of the nucleotides. During cell division, each daughter cell receives a copy of DNA in a process called replication. In the years since the discovery of the structure of DNA, many technologies, including DNA sequencing, have been developed that enable us to better understand DNA and its role in our genomes.
Discovering the Structure of DNA
In the 1950s, Crick and Watson worked together to determine the structure of DNA at the University of Cambridge, England. Other scientists like Linus Pauling and Maurice Wilkins were also actively exploring this field. Pauling previously had discovered the secondary structure of proteins using X-ray crystallography. In Wilkins’ lab, researcher Rosalind Franklin was using X-ray diffraction methods to understand the structure of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of Franklin’s data because Crick had also studied X-ray diffraction. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine. Unfortunately, by then Franklin had died, and Nobel prizes are not awarded posthumously.
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. Base pairing takes place between a purine and pyrimidine on opposite strands, so that A pairs with T, and G pairs with C (suggested by Chargaff’s Rules). Thus, adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. The base pairs are stabilized by hydrogen bonds: adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature; that is, the 3′ end of one strand faces the 5′ end of the other strand. The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside, like the rungs of a ladder. Each base pair is separated from the next base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, ten base pairs are present per turn of the helix. The diameter of the DNA double-helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine and the antiparallel orientation of the two DNA strands can explain the uniform diameter. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves.
DNA Sequencing Techniques
Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process. Using radiolabeled nucleotides also compounded the problem through safety concerns. With currently available technology and automated machines, the process is cheaper, safer, and can be completed in a matter of hours. Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today.
The sequencing method is known as the dideoxy chain termination method. The method is based on the use of chain terminators, the dideoxynucleotides (ddNTPs). The ddNTPSs differ from the deoxynucleotides by the lack of a free 3′ OH group on the five-carbon sugar. If a ddNTP is added to a growing DNA strand, the chain cannot be extended any further because the free 3′ OH group needed to add another nucleotide is not available. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes.
The DNA sample to be sequenced is denatured (separated into two strands by heating it to high temperatures). The DNA is divided into four tubes in which a primer, DNA polymerase, and all four nucleoside triphosphates (A, T, G, and C) are added. In addition, limited quantities of one of the four dideoxynucleoside triphosphates (ddCTP, ddATP, ddGTP, and ddTTP) are added to each tube respectively. The tubes are labeled as A, T, G, and C according to the ddNTP added. For detection purposes, each of the four dideoxynucleotides carries a different fluorescent label. Chain elongation continues until a fluorescent dideoxy nucleotide is incorporated, after which no further elongation takes place. After the reaction is over, electrophoresis is performed. Even a difference in length of a single base can be detected. The sequence is read from a laser scanner that detects the fluorescent marker of each fragment. For his work on DNA sequencing, Sanger received a Nobel Prize in Chemistry in 1980.
Gel electrophoresis is a technique used to separate DNA fragments of different sizes. Usually the gel is made of a chemical called agarose. Agarose powder is added to a buffer and heated. After cooling, the gel solution is poured into a casting tray. Once the gel has solidified, the DNA is loaded on the gel and an electric current is applied. The DNA has a net negative charge and moves from the negative electrode toward the positive electrode. The electric current is applied for sufficient time to let the DNA separate according to size; the smallest fragments will be farthest from the well (where the DNA was loaded), and the heavier molecular weight fragments will be closest to the well. Once the DNA is separated, the gel is stained with a DNA-specific dye for viewing it.
Reflect: Developing Scientific Concepts
It took a great number of scientists, all working in different areas in different locations, to develop an understanding of the structure of DNA over time. Many scientific concepts are developed this way.
Expand: DNA Packing in Cells
Prokaryotes are much simpler than eukaryotes in many of their features. Most prokaryotes contain a single, circular chromosome that is found in an area of the cytoplasm called the nucleoid region.
The size of the genome in one of the most well-studied prokaryotes, E. coli, is 4.6 million base pairs (approximately 1.1 mm, if cut and stretched out). So how does this fit inside a small bacterial cell? The DNA is twisted by what is known as supercoiling. Supercoiling suggests that DNA is either “under-wound” (less than one turn of the helix per ten base pairs) or “over-wound” (more than one turn per ten base pairs) from its normal relaxed state. Some proteins are known to be involved in the supercoiling; other proteins and enzymes such as DNA gyrase help in maintaining the supercoiled structure.
Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a different type of packing strategy to fit their DNA inside the nucleus. At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that are rich in basic amino acids and form an octamer composed of two molecules of each of four different histones. The DNA (remember, it is negatively charged because of the phosphate groups) is wrapped tightly around the histone core. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the “beads on a string” structure. With the help of a fifth histone, a string of nucleosomes is further compacted into a 30-nm fiber, which is the diameter of the structure. Metaphase chromosomes are even further condensed by association with scaffolding proteins. At the metaphase stage, the chromosomes are at their most compact, approximately 700 nm in width.
In interphase, eukaryotic chromosomes have two distinct regions that can be distinguished by staining. The tightly packaged region is known as heterochromatin, and the less dense region is known as euchromatin. Heterochromatin usually contains genes that are not expressed, and is found in the regions of the centromere and telomeres. The euchromatin usually contains genes that are transcribed, with DNA packaged around nucleosomes but not further compacted.
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Which enzyme is only found in prokaryotic organisms?CorrectIncorrect
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What is a purine?CorrectIncorrect
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DNA has a ___________ structure.CorrectIncorrect
Additional Resources and Readings
A Crash Course Biology video covering DNA structure and replication
A video explaining the DNA sequence-reading technique that resulted from Sanger’s work
An animation showing a method of DNA sequencing
Svante Pääbo’s TED talk explaining Neanderthal genome research
- electrophoresistechnique used to separate DNA fragments according to size
License and Citations
Authored and curated by Jill Carson for The TEL Library. CC BY NC SA 4.0
Title: Biology – 14.2 DNA Structure and Sequencing – DNA Sequencing Techniques: Rice University, OpenStax CNX. License: CC BY 4.0
|Dna Biology Medicine||PublicDomainPictures||Pixabay||CC 0|
|Figure 7. compaction of the eukaryotic chromosome||OpenStax||OpenStax||CC BY 4.0|
|Figure 4. Frederick Sanger’s dideoxy chain||OpenStax||OpenStax||CC BY 4.0|
|Figure 3. a double helix structure||OpenStax||OpenStax||CC BY 4.0|
|Figure 2. James Watson, Francis Crick, and Maclyn McCarty||OpenStax||OpenStax||CC BY 4.0|