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Organic Chemistry
Table of Contents
Unit 28 Overview: Biomolecules
28.1 Nucleotides and Nucleic Acids
28.2 Base Pairing in DNA
28.3 Replication of DNA
28.4 Transcription of DNA
28.5 Translation of RNA: Protein Biosynthesis
28.6 DNA Sequencing
28.7 DNA Synthesis
28.8 The Polymerase Chain Reaction

DNA sequencing unravels the genetic code, revealing the order of nucleotides in our DNA. It's like decoding a secret message written in the language of life. This process is crucial for understanding genes, diseases, and evolution.

From basic techniques using restriction enzymes to advanced automated methods, DNA sequencing has come a long way. These tools allow scientists to read entire genomes, opening doors to personalized medicine and groundbreaking discoveries in genetics.

DNA Sequencing

DNA sequencing with restriction enzymes

  • Determines the order of nucleotide bases (A, T, C, G) in a DNA molecule
  • Uses restriction enzymes to cut DNA into smaller, more manageable fragments
    • Recognize specific nucleotide sequences and cleave DNA at those sites (EcoRI, BamHI)
  • Employs Sanger dideoxy method (chain termination method) to sequence DNA fragments
    • Denatures DNA into single strands and mixes with primer, DNA polymerase, and nucleotides (dNTPs)
    • Includes small amount of modified nucleotides called dideoxynucleotides (ddNTPs)
      • Lack 3' hydroxyl group, preventing further elongation of DNA strand
    • Sets up four separate reactions, each containing different ddNTP (ddATP, ddTTP, ddCTP, ddGTP)
    • Extends primer with DNA polymerase, incorporating both dNTPs and ddNTPs
      • Incorporation of ddNTP causes chain termination, resulting in DNA fragments of various lengths
    • Separates fragments by size using gel electrophoresis (agarose gel)
    • Reads sequence from gel, with shortest fragments at bottom and longest at top
  • Relies on complementary base pairing for accurate sequencing results

Automated DNA sequencing process

  • Based on Sanger dideoxy method but uses fluorescent labeling and capillary electrophoresis
    • Labels each ddNTP with different fluorescent dye (FAM, VIC, NED, ROX)
    • Combines four separate reactions into single tube
  • Employs capillary electrophoresis to separate DNA fragments
    • Injects fragments into thin capillary filled with polymer matrix
    • Applies electric field, causing fragments to migrate through capillary
    • Moves shorter fragments faster than longer fragments
  • Excites fragments with laser as they reach end of capillary
    • Detects fluorescent dyes emitting light at different wavelengths with sensor
    • Corresponds order of colored peaks to DNA sequence
  • Offers faster and more accurate sequencing than manual methods
    • Sequences up to 1,000 bases per run with 99.9% accuracy
    • Processes multiple samples simultaneously using 96-well plates (high-throughput sequencing)

Advanced DNA Sequencing Techniques

  • Polymerase chain reaction (PCR) amplifies specific DNA sequences for sequencing
  • Next-generation sequencing (NGS) enables high-throughput, parallel sequencing of multiple DNA fragments
  • Bioinformatics tools analyze and interpret large-scale sequencing data
  • Genome assembly techniques reconstruct complete genomic sequences from shorter sequenced fragments

Human genes vs human proteins

  • Human genome contains approximately 20,000-25,000 protein-coding genes
  • Human proteome (complete set of proteins) estimated to be much larger, with over 1 million different proteins
  • Explains discrepancy between number of genes and proteins by several factors:
    1. Alternative splicing: Produces multiple mRNA variants from single gene, leading to different protein isoforms
      • Includes or excludes exons in various combinations, creating diverse mRNA molecules (CD44, DSCAM)
    2. Post-translational modifications: Alters proteins after translation, such as phosphorylation, glycosylation, or acetylation
      • Modifies protein function, stability, or localization (p53, histones)
    3. Proteolytic cleavage: Synthesizes some proteins as inactive precursors requiring cleavage to become active
      • Generates multiple functional proteins from single precursor (insulin, enkephalins)
    4. Protein complexes: Forms complexes with other proteins, creating new functional units with distinct properties
      • Combines proteins to form multisubunit enzymes or signaling complexes (hemoglobin, proteasome)

Key Terms to Review (52)

Sanger dideoxy method: The Sanger dideoxy method is a technique used for DNA sequencing by selectively incorporating chain-terminating dideoxynucleotides during DNA synthesis. It relies on the use of normal nucleotides and modified versions that prevent further extension, allowing the determination of the DNA sequence.
Posttranslational modification: Posttranslational modification is a biochemical process where proteins undergo changes after their synthesis, involving the addition or removal of chemical groups. These modifications can alter a protein's function, activity, stability, and location within the cell.
Restriction endonucleases: Restriction endonucleases are enzymes that recognize specific sequences of DNA and cut the DNA at or near these sites. They are essential tools in molecular biology for DNA manipulation, including cloning and DNA sequencing.
Hemoglobin: Hemoglobin is the iron-rich protein found in red blood cells that is responsible for transporting oxygen from the lungs to the body's tissues and carbon dioxide from the tissues back to the lungs. It is a crucial component in the processes of protein structure and DNA sequencing.
DNA Polymerase: DNA polymerase is a crucial enzyme responsible for the replication and repair of DNA molecules. It plays a vital role in ensuring the accurate duplication of genetic information during cell division, as well as the maintenance of genomic integrity through DNA repair processes.
Insulin: Insulin is a hormone produced by the pancreas that regulates blood sugar levels by facilitating the uptake and utilization of glucose by cells. It is a critical component in the metabolic processes of protein structure, DNA sequencing, fatty acid biosynthesis, and carbohydrate metabolism through gluconeogenesis.
Complementary Base Pairing: Complementary base pairing is a fundamental principle in molecular biology, where specific nucleic acid bases form pairs with each other to maintain the structure and function of genetic material, such as DNA and RNA. This concept is central to understanding the storage, replication, and expression of genetic information across various biological topics.
MRNA: mRNA, or messenger RNA, is a single-stranded molecule that carries the genetic instructions from the nucleus to the ribosomes in the cytoplasm, where protein synthesis occurs. It serves as a template for the production of specific proteins within the cell.
Fluorescent Labeling: Fluorescent labeling is a technique used in molecular biology and biochemistry to visualize and track specific biomolecules, such as DNA, proteins, or cells, by attaching fluorescent dyes or markers to them. These fluorescent labels emit light when excited by a specific wavelength of light, allowing the labeled biomolecules to be detected and studied using specialized imaging equipment.
Proteasome: The proteasome is a large, barrel-shaped protein complex found in the cytoplasm and nucleus of eukaryotic cells. It is responsible for the degradation and recycling of unwanted or damaged proteins, playing a crucial role in cellular protein homeostasis and regulation.
Bioinformatics: Bioinformatics is an interdisciplinary field that combines biology, computer science, and information technology to analyze and interpret biological data, particularly genetic and genomic information. It plays a crucial role in DNA sequencing and understanding the structure, function, and evolution of living organisms.
DdATP: ddATP, or dideoxynucleotide triphosphate, is a modified nucleotide used in the DNA sequencing process known as the Sanger method. It is an analog of the natural nucleotide dATP, with the key difference being the absence of a 3' hydroxyl group on the deoxyribose sugar.
Enkephalins: Enkephalins are a class of endogenous opioid peptides found in the brain and nervous system that function as neurotransmitters and neuromodulators, playing a role in pain perception, mood regulation, and other physiological processes.
Alternative Splicing: Alternative splicing is a process in gene expression where a single gene can produce multiple different mRNA transcripts by selectively including or excluding different segments of the gene's coding sequence. This allows a single gene to encode for multiple distinct protein isoforms, greatly expanding the functional diversity of the proteome from a limited number of genes.
DNA Sequencing: DNA sequencing is the process of determining the precise order of the four chemical building blocks (adenine, guanine, cytosine, and thymine) that make up a DNA molecule. This technique allows scientists to read the genetic code and gather information about the structure and function of genes and genomes.
DNTPs: dNTPs, or deoxyribonucleotide triphosphates, are the building blocks of DNA. They are the four nucleotides (adenine, guanine, cytosine, and thymine) that are used by DNA polymerase to replicate and synthesize new DNA strands during processes like DNA sequencing and the Polymerase Chain Reaction (PCR).
DdNTPs: ddNTPs, or dideoxynucleotide triphosphates, are synthetic nucleotides used in the DNA sequencing process known as the Sanger method. These modified nucleotides lack a 3' hydroxyl group, which prevents the formation of a phosphodiester bond, effectively terminating DNA chain elongation.
DdTTP: ddTTP, or dideoxynucleotide triphosphate, is a modified nucleotide used in the DNA sequencing method known as the Sanger sequencing technique. It plays a crucial role in the process of determining the precise order of nucleotides in a DNA molecule.
VIC: VIC, or Variant Identification and Calling, is a crucial process in the field of DNA sequencing that involves the detection and characterization of genetic variations within a DNA sequence. It is a fundamental step in understanding the genetic makeup of an individual or a population, and it has important applications in areas such as medical diagnostics, population genetics, and evolutionary studies.
ROX: ROX, or ROX Fluorescent Dye, is a type of fluorescent dye commonly used in DNA sequencing techniques. It is a key component in the process of identifying and analyzing the nucleotide sequence of DNA samples.
Human Genome: The human genome refers to the complete set of genetic information encoded in the DNA of a human individual. It encompasses all the genes and non-coding DNA sequences that make up the human chromosomes, providing the essential instructions for the development, function, and reproduction of the human organism.
DSCAM: DSCAM (Down Syndrome Cell Adhesion Molecule) is a gene that encodes a protein involved in the development and function of the nervous system. It plays a crucial role in neuronal wiring and connectivity, particularly in the context of DNA sequencing techniques used to study genetic variations and their impact on neurological disorders.
Exons: Exons are the coding sequences within a gene that are retained in the mature messenger RNA (mRNA) molecule and ultimately translated into the amino acid sequence of a protein. They represent the functional, protein-coding regions of a gene, in contrast to the non-coding introns that are removed during the process of RNA splicing.
Phosphorylation: Phosphorylation is a fundamental biochemical process in which a phosphate group is added to a molecule, often a protein, altering its function or activity. This process is crucial in various cellular processes, including DNA sequencing, carbohydrate catabolism, and the overall regulation of biological chemistry.
CD44: CD44 is a cell surface glycoprotein that functions as a receptor for hyaluronic acid and other extracellular matrix components. It is involved in various cellular processes, including cell-cell and cell-matrix interactions, cell migration, and signal transduction.
EcoRI: EcoRI is a type of restriction enzyme, a protein that can recognize and cleave specific DNA sequences, allowing for the manipulation and analysis of genetic material. It is a crucial tool in the field of DNA sequencing and genetic engineering.
FAM: FAM, short for Fluorescein Amidite, is a fluorescent dye commonly used in DNA sequencing techniques. It is a derivative of the fluorescein molecule that can be covalently attached to DNA or RNA oligonucleotides, enabling the detection and analysis of genetic sequences during the DNA sequencing process.
DdCTP: ddCTP, or dideoxycytidine triphosphate, is a modified nucleotide used in the DNA sequencing method known as the Sanger sequencing technique. It plays a crucial role in determining the sequence of DNA molecules by acting as a chain terminator during DNA synthesis.
Protein Complexes: Protein complexes are assemblies of multiple protein molecules that come together to perform a specific biological function. They are essential for a wide range of cellular processes, including DNA replication, transcription, and translation, as well as signaling pathways and metabolic reactions.
NED: NED, or Next-Generation Exome Sequencing, is a powerful DNA sequencing technique that allows for the rapid and efficient analysis of the protein-coding regions of the human genome, known as the exome. This technology has revolutionized the field of genomics and has become a crucial tool in the diagnosis and understanding of genetic disorders.
Next-Generation Sequencing (NGS): Next-Generation Sequencing (NGS) is a high-throughput DNA sequencing technology that enables rapid and cost-effective analysis of genetic information. It has revolutionized the field of genomics by allowing for the simultaneous sequencing of millions of DNA fragments in a single run, providing a comprehensive view of an organism's genetic makeup.
Polymerase Chain Reaction (PCR): Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to exponentially amplify specific DNA sequences from a small amount of starting material. It is a fundamental tool in DNA sequencing, synthesis, and analysis, with applications across various fields of biology and medicine.
Genome Assembly: Genome assembly is the process of reconstructing the complete DNA sequence of an organism from short, overlapping DNA fragments obtained through DNA sequencing. It involves computationally piecing together these fragmented sequences to create a contiguous representation of the original genome.
Acetylation: Acetylation is a chemical process that involves the addition of an acetyl group (-COCH3) to a molecule, typically a protein or DNA. This modification can have significant effects on the structure, function, and regulation of the target molecule.
Restriction Enzymes: Restriction enzymes are specialized proteins found in bacteria and archaea that can recognize and cut specific DNA sequences, known as restriction sites. These enzymes act as molecular scissors, cleaving DNA molecules at precise locations, which is a crucial process in DNA sequencing and various genetic engineering techniques.
Nucleotide Bases: Nucleotide bases are the fundamental building blocks of nucleic acids, such as DNA and RNA. They consist of a nitrogenous base, a pentose sugar, and a phosphate group, and are responsible for storing and transmitting genetic information within living organisms.
Chain Termination Method: The chain termination method, also known as the Sanger sequencing method, is a DNA sequencing technique that allows for the determination of the precise order of nucleotides (A, T, C, and G) within a DNA molecule. This method relies on the controlled termination of DNA synthesis to generate a set of DNA fragments of varying lengths, which can then be analyzed to decipher the DNA sequence.
Dideoxynucleotides: Dideoxynucleotides are synthetic DNA building blocks that lack the 3' hydroxyl group, making them unable to form the 3'-5' phosphodiester bonds required for DNA chain elongation. This unique property allows them to be used in DNA sequencing techniques to precisely determine the sequence of nucleotides in a DNA molecule.
Agarose Gel: Agarose gel is a porous, semisolid matrix used in various biotechnological and biochemical applications, particularly in the context of DNA sequencing. It is derived from a polysaccharide extracted from red algae and is widely used as a separation and purification medium for DNA, RNA, and proteins.
Capillary Electrophoresis: Capillary electrophoresis is an analytical technique used to separate and analyze complex mixtures of molecules, such as DNA, proteins, or small organic compounds, based on their differential migration through a narrow, charged capillary under the influence of an applied electric field.
High-Throughput Sequencing: High-throughput sequencing, also known as next-generation sequencing (NGS), is a DNA sequencing technology that allows for the rapid and efficient analysis of large amounts of genetic material. It enables the simultaneous sequencing of millions or billions of DNA fragments, providing a comprehensive and detailed view of an organism's genome or transcriptome.
Polymer Matrix: A polymer matrix refers to a composite material composed of a continuous phase of polymer material that surrounds and binds together a dispersed phase of another material, such as fibers, particles, or other reinforcements. The polymer matrix acts as the structural foundation and provides mechanical integrity to the overall composite system.
Histones: Histones are a group of small, positively charged proteins that play a crucial role in the organization and regulation of DNA within the eukaryotic cell nucleus. They act as spools around which DNA winds, forming the basic unit of chromatin called the nucleosome, which is essential for DNA sequencing and other genetic processes.
Proteolytic Cleavage: Proteolytic cleavage is the process by which a protein is broken down into smaller peptide fragments or individual amino acids through the action of enzymes called proteases. This process is crucial for various biological functions, including protein digestion, activation of enzymes and hormones, and regulation of cellular processes.
Glycosylation: Glycosylation is the enzymatic process of adding glycan (carbohydrate) chains to proteins, lipids, or other organic molecules. This post-translational modification is crucial for the proper folding, stability, and function of many biomolecules, including those involved in DNA sequencing.
Human Proteome: The human proteome refers to the complete set of proteins expressed by the human genome. It represents the diverse array of proteins that are involved in the structure, function, and regulation of the human body's tissues and organs.
P53: p53 is a tumor suppressor protein that plays a crucial role in regulating the cell cycle, DNA repair, and apoptosis (programmed cell death). It is often referred to as the 'guardian of the genome' due to its ability to prevent the propagation of cells with damaged DNA, which could otherwise lead to cancer development.
BamHI: BamHI is a type II restriction endonuclease enzyme that recognizes and cleaves a specific DNA sequence, making it a crucial tool in DNA sequencing and genetic engineering.
Gel Electrophoresis: Gel electrophoresis is a laboratory technique used to separate and analyze DNA, RNA, or protein molecules based on their size and electrical charge. It is a crucial tool in the field of DNA sequencing, allowing researchers to visualize and study the genetic material of organisms.
Sanger Dideoxy Method: The Sanger dideoxy method, also known as the chain-termination method, is a DNA sequencing technique developed by Frederick Sanger in the 1970s. It is a widely used method for determining the precise order of nucleotides (A, T, C, and G) in a DNA molecule, which is crucial for understanding genetic information and analyzing DNA sequences.
DdGTP: ddGTP, or dideoxynucleotide triphosphate, is a modified nucleotide used in the Sanger DNA sequencing method. It serves as a chain terminator during DNA synthesis, allowing for the determination of the DNA sequence by identifying the terminal nucleotide in each synthesized fragment.
Post-Translational Modifications: Post-translational modifications (PTMs) are chemical changes made to a protein after it has been synthesized, which can alter the protein's structure, function, and interactions. These modifications occur in the cell following the translation of mRNA into a polypeptide chain, and they are crucial for regulating protein activity, stability, and localization within the cell.