14.2 DNA Structure and Sequencing
3 min read•june 14, 2024
is the blueprint of life, containing instructions for every living organism. Its structure, composed of nucleotides and held together by , allows for accurate and of genetic information.
Understanding DNA's structure is crucial for grasping how genetic information is stored and passed on. This knowledge forms the foundation for advanced techniques like DNA sequencing and studying how DNA is packaged in different organisms.
DNA Structure
Illustrate the double helix structure of DNA, including its nucleotide components and base pairing
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- DNA () double-stranded helical molecule
- Two chains coil around each other forming right-handed double helix
- Strands run in opposite directions (antiparallel)
- One strand runs 5' to 3' while other runs 3' to 5'
- Polynucleotide chain composed of nucleotides
- consists of three components
- (, , , )
- sugar
- Phosphate group
- Nucleotides joined by between 5' phosphate group of one nucleotide and 3' hydroxyl group of adjacent nucleotide
- consists of three components
- Base pairing occurs between two strands of DNA
- Adenine (A) pairs with thymine (T) via two
- Guanine (G) pairs with cytosine (C) via three hydrogen bonds
- Base pairs stabilized by hydrogen bonding and
- Complementary nature of base pairing ensures specificity and accuracy of DNA replication and transcription (protein synthesis)
- allows for faithful DNA replication and transcription
DNA Sequencing
Steps in Sanger sequencing
- DNA : Double-stranded DNA template separated into single strands by heating
- : Short oligonucleotide primer complementary to specific region of template DNA added and binds to single-stranded DNA
- Extension and termination
- extends primer synthesizing new complementary strand
- Four separate reactions set up each containing all four triphosphates () and small amount of one of four dideoxynucleotide triphosphates ()
- When ddNTP incorporated chain elongation terminated resulting in DNA fragments of varying lengths
- : Terminated DNA fragments separated by size using polyacrylamide
- Visualization and sequence determination: DNA fragments visualized using or fluorescence detection and sequence read from gel
- instrumental in advancing understanding of genomes and genetic variation
- Used to sequence entire genomes including human genome
- Still used for targeted sequencing of specific regions of interest and validating results from other sequencing technologies ()
- often used to amplify DNA samples before sequencing
Advanced DNA Sequencing Techniques
- has revolutionized our understanding of genetics and evolution
- High-throughput sequencing technologies enable rapid and cost-effective sequencing of entire genomes
- , where each new DNA molecule contains one original strand and one newly synthesized strand, is fundamental to DNA replication and sequencing techniques
DNA Packaging
DNA packaging in eukaryotes vs prokaryotes
- Prokaryotic DNA organization
- Prokaryotic cells (bacteria, archaea) have single circular chromosome located in region
- DNA not enclosed within membrane-bound nucleus
- Prokaryotic DNA associated with proteins called (NAPs) which help compact and organize DNA
- Prokaryotic DNA not extensively packaged as genome relatively small and cell lacks nucleus
- Eukaryotic DNA organization
- Eukaryotic cells have multiple linear chromosomes contained within membrane-bound nucleus
- Eukaryotic DNA extensively packaged to fit within nucleus and regulate gene expression
- Packaging of eukaryotic DNA involves several levels of organization
- : DNA wrapped around protein octamers to form nucleosomes basic unit of
- Chromatin fibers: Nucleosomes further coiled and compacted to form chromatin fibers
- Higher-order structures: Chromatin fibers folded and looped to form higher-order structures such as chromatin loops and (TADs)
- Chromosomes: During cell division chromatin further condensed to form compact chromosomes
- Packaging of eukaryotic DNA allows for tight regulation of gene expression and proper segregation of chromosomes during cell division (, )
Key Terms to Review (46)
Adenine: Adenine is one of the four primary nitrogenous bases found in DNA and RNA, specifically classified as a purine. It plays a crucial role in the storage and transfer of genetic information and energy in cells. In nucleic acids, adenine pairs with thymine in DNA and uracil in RNA, forming the rungs of the molecular ladder that composes the double helix structure.
Antiparallel strands: Antiparallel strands refer to the arrangement of two strands of nucleotides in a DNA molecule where one strand runs in the 5' to 3' direction and the other strand runs in the 3' to 5' direction. This unique orientation is crucial for the structural stability of DNA and is essential during processes like replication and transcription, where enzymes read the template strand in a specific direction.
Autoradiography: Autoradiography is a technique used to visualize the distribution of radioactively labeled molecules, particularly DNA or RNA, within a sample. This method allows researchers to detect specific sequences by exposing the sample to a photographic film or a phosphor screen, capturing the emitted radiation. Autoradiography plays a critical role in studying DNA structure and sequencing, as it provides insight into the location and abundance of nucleic acids in various biological contexts.
Base pairing: Base pairing refers to the specific hydrogen bonding between nitrogenous bases in nucleic acids, such as DNA and RNA, that ensures the accurate replication and transcription of genetic information. In DNA, adenine pairs with thymine (A-T) and cytosine pairs with guanine (C-G), while in RNA, adenine pairs with uracil (A-U) instead of thymine. This complementary nature of base pairing is fundamental to the structure of DNA and its ability to store and transmit genetic information.
Chromatin: Chromatin is a complex of DNA and protein found in the nucleus of eukaryotic cells that serves to package DNA into a more compact form, allowing for efficient regulation of gene expression and DNA replication. It plays a crucial role in determining the accessibility of DNA for transcription, replication, and repair processes, impacting how genes are expressed and regulated throughout the cell cycle.
Complementary base pairing: Complementary base pairing is the specific hydrogen bonding between nucleotide bases in DNA and RNA, where adenine pairs with thymine (or uracil in RNA) and cytosine pairs with guanine. This pairing is crucial for maintaining the double helical structure of DNA, ensuring accurate replication, and facilitating the process of transcription. By forming stable bonds between complementary bases, this mechanism supports genetic fidelity and proper gene expression.
Complementary DNA (cDNA) libraries: cDNA libraries are collections of complementary DNA (cDNA) sequences synthesized from mRNA templates. They are used to study gene expression and identify coding regions in genomics research.
Cytosine: Cytosine is one of the four primary nitrogenous bases found in nucleic acids, specifically DNA and RNA. It pairs with guanine through three hydrogen bonds in DNA, playing a critical role in the structure and function of genetic material, as well as the processes of transcription and replication.
DdNTPs: ddNTPs, or dideoxynucleotide triphosphates, are modified nucleotides used in DNA sequencing techniques, such as Sanger sequencing. They are similar to regular deoxynucleotide triphosphates (dNTPs) but lack a hydroxyl group on the 3' carbon of the sugar, which prevents further elongation of the DNA strand once incorporated. This property is crucial for generating DNA fragments of varying lengths that can be analyzed to determine the sequence of nucleotides in a given DNA molecule.
Denaturation: Denaturation is the process by which a protein loses its native structure and function due to external stress, such as heat or chemicals. This structural change is often irreversible and affects the protein's biological activity.
Denaturation: Denaturation refers to the process where proteins or nucleic acids lose their native structure due to the disruption of weak interactions, resulting in the loss of their biological function. This process can occur due to various factors such as extreme temperature changes, pH shifts, or exposure to certain chemicals, which can break down the intricate folding and bonding that maintains the specific shape necessary for functionality.
Deoxynucleotide: Deoxynucleotide is a molecule consisting of a nitrogenous base, a deoxyribose sugar, and one or more phosphate groups. It is the basic building block of DNA.
Deoxyribonucleic acid: Deoxyribonucleic acid, commonly known as DNA, is the molecule that carries the genetic instructions for the development, functioning, growth, and reproduction of all known living organisms and many viruses. DNA is structured as a double helix composed of two long strands of nucleotides, which include a sugar (deoxyribose), a phosphate group, and nitrogenous bases. This unique structure enables DNA to store and transmit genetic information essential for life.
Deoxyribose: Deoxyribose is a five-carbon sugar that is a crucial component of DNA (deoxyribonucleic acid), forming part of its backbone along with phosphate groups. This sugar distinguishes DNA from RNA (ribonucleic acid) by lacking one oxygen atom, which affects the stability and structure of the nucleic acids. The absence of this oxygen atom in deoxyribose contributes to the double-helix structure of DNA and plays an important role in the genetic coding and information storage within living organisms.
DNA: DNA, or deoxyribonucleic acid, is the hereditary material in nearly all living organisms, encoding the genetic instructions that govern the development, functioning, growth, and reproduction of cells. This molecule is central to many biological processes, linking the concepts of genetic inheritance to molecular biology and the chemistry of life.
DNA polymerase: DNA polymerase is an essential enzyme responsible for synthesizing new strands of DNA by adding nucleotides to a growing DNA chain during DNA replication. It plays a critical role in ensuring the accuracy and fidelity of DNA replication, which is fundamental to cell division, gene expression, and the overall maintenance of genetic information.
DNTPs: dNTPs, or deoxynucleotide triphosphates, are the building blocks of DNA, consisting of a deoxyribose sugar, a nitrogenous base (adenine, thymine, cytosine, or guanine), and three phosphate groups. These molecules play a crucial role in DNA synthesis and sequencing, as they are incorporated into the growing DNA strand during replication and serve as substrates for various enzymatic reactions involved in DNA manipulation.
Double helix: The double helix is the structural formation of DNA, consisting of two intertwined strands that resemble a twisted ladder. This unique structure is crucial for the stability and functionality of DNA, allowing it to store genetic information efficiently while providing the mechanism for replication and transcription.
Gel electrophoresis: Gel electrophoresis is a technique used to separate DNA, RNA, or proteins based on their size and charge. It utilizes an electric field to move the molecules through a gel matrix.
Gel electrophoresis: Gel electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins based on their size and charge by applying an electric field to a gel matrix. This method relies on the fact that smaller molecules migrate faster through the gel than larger ones, allowing for the analysis of genetic material and the visualization of biomolecules, which is crucial for understanding genetic sequences, cloning, and genetic mapping.
Genome sequencing: Genome sequencing is the process of determining the complete DNA sequence of an organism's genome, which includes all of its genes and non-coding sequences. This technique provides critical insights into the genetic makeup of organisms, enabling scientists to understand genetic variations, evolutionary relationships, and the basis of diseases.
Guanine: Guanine is one of the four primary nitrogenous bases found in nucleic acids, specifically DNA and RNA. It plays a critical role in the storage and transmission of genetic information and pairs with cytosine in the structure of DNA, contributing to the double helix's stability. This base is essential for protein synthesis and other cellular functions, making it a vital component in the molecular biology of all living organisms.
Histone: Histones are highly alkaline proteins that play a crucial role in the organization and packaging of DNA within the nucleus of eukaryotic cells. By forming nucleosomes, histones help condense long strands of DNA into a more compact structure, facilitating efficient cell division, protecting DNA integrity, and regulating gene expression.
Histone acetylation: Histone acetylation is the addition of an acetyl group to histone proteins, leading to a more relaxed chromatin structure and increased gene expression. This process is crucial for regulating access to DNA by transcriptional machinery.
Hydrogen Bonds: Hydrogen bonds are weak interactions that occur between a hydrogen atom covalently bonded to an electronegative atom and another electronegative atom. These bonds play a crucial role in stabilizing the structure of nucleic acids, particularly DNA, by forming connections between complementary base pairs, which is essential for the integrity and function of genetic material.
Hydrophobic interactions: Hydrophobic interactions are the forces that drive non-polar molecules or regions of molecules to aggregate in aqueous environments, minimizing their exposure to water. These interactions play a critical role in the structure and stability of biological macromolecules like proteins and nucleic acids, including DNA, where non-polar bases tend to cluster away from water, leading to the formation of distinct structures essential for their function.
Meiosis: Meiosis is a specialized form of cell division that reduces the chromosome number by half, resulting in the production of four genetically diverse gametes, or sex cells. This process is crucial for sexual reproduction, as it ensures genetic diversity and maintains the species' chromosome number across generations.
Mitosis: Mitosis is the process of cell division that results in two genetically identical daughter cells, each containing the same number of chromosomes as the original cell. This process is essential for growth, development, and tissue repair in multicellular organisms, linking it to various biological concepts including cellular organization and reproduction.
Next-generation sequencing: Next-generation sequencing (NGS) is a revolutionary DNA sequencing technology that enables the rapid sequencing of large amounts of DNA by simultaneously analyzing millions of fragments. This technology has transformed genomics by allowing researchers to sequence entire genomes quickly and at a lower cost, thereby facilitating advancements in genetics, personalized medicine, and biological research.
Nitrogenous Base: A nitrogenous base is a molecular component of nucleic acids that contains nitrogen and acts as a fundamental building block for DNA and RNA. These bases are essential for encoding genetic information, pairing through hydrogen bonds, and contributing to the overall structure of nucleic acids. In DNA, there are four primary nitrogenous bases—adenine, thymine, cytosine, and guanine—while RNA contains adenine, uracil, cytosine, and guanine.
Nucleoid: The nucleoid is a region within prokaryotic cells where the cell's circular DNA is located, playing a crucial role in cellular processes such as replication and gene expression. Unlike eukaryotic cells, which have a defined nucleus, prokaryotic cells have their genetic material concentrated in this non-membrane-bound area, allowing for efficient regulation of DNA functions in processes like cell division and adaptation.
Nucleoid-associated proteins: Nucleoid-associated proteins (NAPs) are a group of proteins that play a critical role in the organization and compactness of bacterial DNA within the nucleoid region. These proteins help to bend, loop, and fold the DNA, facilitating its efficient packing and regulation of gene expression. NAPs are essential for maintaining the structure of chromosomal DNA in prokaryotes, impacting processes such as replication and transcription.
Nucleosomes: Nucleosomes are the fundamental units of chromatin, consisting of a segment of DNA wrapped around a core of histone proteins. This structure plays a crucial role in the packaging of DNA into a compact, organized form, which is essential for efficient gene regulation and DNA replication. Nucleosomes also contribute to the overall stability of the DNA molecule and help control access to genetic information.
Nucleotide: A nucleotide is the basic building block of nucleic acids, such as DNA and RNA, composed of three components: a phosphate group, a five-carbon sugar, and a nitrogenous base. These components work together to form the structure of DNA and RNA, enabling the storage and transmission of genetic information.
Phosphodiester bonds: Phosphodiester bonds are covalent bonds that link the phosphate group of one nucleotide to the hydroxyl group on the sugar of another nucleotide, forming the backbone of DNA and RNA. These bonds are crucial for maintaining the structural integrity of nucleic acids, allowing them to hold genetic information securely and facilitating processes like replication and transcription.
Polymerase chain reaction (PCR): Polymerase chain reaction (PCR) is a laboratory technique used to amplify specific segments of DNA, making millions of copies from a small initial sample. This process relies on repeated cycles of denaturation, annealing, and extension to exponentially increase the amount of target DNA, making it a vital tool in various applications such as genetic testing, forensics, and research.
Polynucleotide: A polynucleotide is a long chain of nucleotides linked together by phosphodiester bonds, forming the backbone of nucleic acids such as DNA and RNA. These chains are crucial for genetic information storage and transfer, with each nucleotide consisting of a sugar, a phosphate group, and a nitrogenous base. The sequence of these nucleotides encodes genetic information, influencing everything from traits to cellular functions.
Post-transcriptional: Post-transcriptional regulation refers to the control of gene expression at the RNA level, after transcription has occurred. This can include processes such as RNA splicing, editing, transport, and degradation.
Primer annealing: Primer annealing is the process where short sequences of nucleotides, known as primers, bind to complementary sequences on a single-stranded DNA template during the initial stages of DNA replication or polymerase chain reaction (PCR). This binding is crucial because it provides a starting point for DNA synthesis, allowing enzymes like DNA polymerase to extend the primer and replicate the DNA strand.
Replication: Replication is the process by which a cell duplicates its DNA, ensuring that each new cell receives an exact copy of the genetic material during cell division. This vital process is critical for maintaining genetic fidelity, allowing for proper functioning and inheritance of traits in organisms. Furthermore, replication serves as a foundation for understanding how genetic information is passed down, as well as how viruses replicate within their host cells.
Sanger sequencing: Sanger sequencing is a method used to determine the precise order of nucleotides in a DNA molecule by incorporating chain-terminating dideoxynucleotides during DNA replication. This technique, developed by Frederick Sanger in the 1970s, allows for the accurate sequencing of both short and long stretches of DNA, making it a foundational tool in genetics and molecular biology for analyzing DNA structure and sequencing.
Semiconservative replication: Semiconservative replication is the process by which DNA makes copies of itself, where each new double helix consists of one original strand and one newly synthesized strand. This method ensures that the genetic information is preserved through generations, allowing for accurate transmission of hereditary traits.
Thymine: Thymine is one of the four nucleotide bases found in DNA, represented by the letter 'T'. It pairs with adenine (A) through two hydrogen bonds, forming the rungs of the DNA ladder structure. Thymine's presence is critical for the stability and integrity of DNA, influencing processes such as base pairing during DNA replication.
Topologically associating domains: Topologically associating domains (TADs) are regions of the genome that interact more frequently with themselves than with other regions, creating a three-dimensional structure that organizes the genome. This organization is important for gene regulation, as it allows for the formation of regulatory landscapes that can influence transcription and other genomic functions.
Transcription: Transcription is the biological process where the DNA sequence of a gene is copied into RNA. This process is essential for gene expression, as it allows the genetic information stored in DNA to be transferred to messenger RNA (mRNA), which then guides protein synthesis. It serves as the first step in expressing genes, linking the genetic code found in DNA to the production of proteins necessary for cellular functions.
Whole-genome sequencing: Whole-genome sequencing is the process of determining the complete DNA sequence of an organism's genome at a single time. It provides comprehensive information about genetic variation and can be used in research, diagnostics, and personalized medicine.