28.7 DNA Synthesis
3 min read•may 7, 2024
DNA synthesis is a crucial process in molecular biology, allowing scientists to create custom genetic sequences. This technique uses a step-by-step approach, adding one at a time to build specific DNA strands. It's like constructing a genetic Lego set, piece by piece.
The process involves protecting and deprotecting various chemical groups, coupling nucleotides, and carefully controlling reactions. Understanding DNA synthesis helps us grasp how researchers can manipulate genetic material for various applications, from studying gene function to developing new therapies.
DNA Synthesis
Steps in automated DNA synthesis
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- Solid support
- Controlled pore glass () beads commonly used as solid support
- First nucleoside attached to support via linker at 3' end, providing starting point for synthesis
- Detritylation (Deprotection)
- Removal of 5'-dimethoxytrityl () protecting group using acid ()
- Exposes 5'-hydroxyl group for next coupling step, allowing addition of next nucleotide
- Coupling
- Activated monomer added, coupling to exposed 5'-hydroxyl group
- used as activator, protonating phosphoramidite to facilitate coupling reaction
- Forms between nucleotides, linking them together
- Capping
- Unreacted 5'-hydroxyl groups acetylated using and
- Prevents unreacted groups from participating in subsequent coupling steps, reducing mutation risk
- Oxidation
- solution (iodine, water, ) used to oxidize to
- Stabilizes internucleotide linkage, preventing unwanted cleavage and ensuring stability
- Cycle repetition
- Steps repeated, adding one nucleotide per cycle, until desired sequence obtained
- Allows for controlled synthesis of with specific sequence
- Cleavage and deprotection
- Oligonucleotide cleaved from solid support using ammonia, releasing synthesized DNA
- Protecting groups (base and phosphate) removed simultaneously during ammonia treatment
Protection of reactive groups
- 5'-Hydroxyl group of growing oligonucleotide protected with DMT
- Prevents unwanted side reactions and ensures controlled growth by blocking reactive site
- Allows for selective deprotection and coupling at 5' end during each synthesis cycle
- Exocyclic amino groups of nucleobases (adenine, cytosine, guanine) protected with (A, C) or (G) groups
- Prevents side reactions by blocking reactive amino groups on bases
- Improves solubility of protected nucleotides, facilitating synthesis process
- Phosphate groups protected with groups
- Prevents unwanted side reactions by blocking reactive phosphate groups
- Improves solubility of protected nucleotides, enhancing synthesis efficiency
Reagents for DNA synthesis steps
- Coupling step
- Phosphoramidite monomers: Building blocks for oligonucleotide synthesis, containing protected nucleobases
- Tetrazole: Activator that protonates phosphoramidite, facilitating coupling reaction between monomers
- Oxidation step
- Iodine solution (iodine, water, pyridine): Oxidizes triester to phosphate triester, stabilizing internucleotide linkage
- Cleavage step
- Concentrated ammonia solution: Cleaves oligonucleotide from solid support and removes base and phosphate protecting groups simultaneously
DNA Structure and Amplification
- Nucleotides: Building blocks of DNA, consisting of a sugar, phosphate group, and nitrogenous base
- : Specific hydrogen bonding between complementary base pairs (A-T and G-C) in DNA double helix
- : Technique for rapid amplification of specific DNA sequences using thermal cycling and DNA polymerase
Key Terms to Review (22)
2-cyanoethyl: The 2-cyanoethyl group is a functional group commonly used in the synthesis of DNA. It consists of a two-carbon chain with a cyano (nitrile) group attached to one of the carbon atoms, providing a protective group for the hydroxyl group during DNA synthesis.
Acetic Anhydride: Acetic anhydride is a colorless, volatile liquid that is used as a reagent in organic synthesis, particularly in the formation of carboxylic acid derivatives. It is a key compound in various chemical reactions and processes, including those related to carboxylic acid derivatives, acid anhydrides, and DNA synthesis.
Benzoyl: Benzoyl is a functional group consisting of a benzene ring attached to a carbonyl (C=O) group. It is commonly found in various organic compounds and plays a crucial role in DNA synthesis.
CPG: CPG, or Cyclic Guanosine Monophosphate, is a crucial second messenger molecule that plays a vital role in DNA synthesis and various cellular processes. It is derived from the nucleotide guanosine triphosphate (GTP) and acts as a signaling molecule, regulating a wide range of physiological functions within the cell.
Deoxyribose: Deoxyribose is a monosaccharide, a type of simple sugar, that serves as the primary structural component of the backbone of deoxyribonucleic acid (DNA) molecules. It is an essential building block for the genetic material found in all living organisms.
DMT: DMT, or N,N-Dimethyltryptamine, is a naturally occurring psychedelic compound found in various plants and animals. It is considered one of the most potent hallucinogens and is known for its ability to produce intense, short-lived psychedelic experiences. In the context of DNA synthesis, DMT is not directly involved, but its unique properties and interactions with the human body can provide insights into the broader understanding of biological processes.
DMT is classified as a tryptamine, a class of compounds that share a common chemical structure. It is structurally similar to the neurotransmitter serotonin and can interact with serotonin receptors in the brain, leading to its profound psychoactive effects.
Iodine: Iodine is a chemical element that is essential for the proper functioning of the thyroid gland, which regulates metabolism and other important bodily processes. It is a key component of the thyroid hormones thyroxine (T4) and triiodothyronine (T3), which are responsible for controlling the body's energy production and utilization.
Isobutyryl: Isobutyryl is a functional group in organic chemistry consisting of a carbonyl carbon atom bonded to a branched alkyl group with three carbon atoms. It is commonly encountered in the context of DNA synthesis, where it plays a crucial role in the structure and function of DNA molecules.
N-methylimidazole: N-methylimidazole is a heterocyclic organic compound that consists of an imidazole ring with a methyl group attached to the nitrogen atom. It is an important intermediate in the synthesis of various pharmaceuticals and other chemical compounds.
Nucleotide: A nucleotide is the basic structural unit of nucleic acids, such as DNA and RNA. It consists of a nitrogenous base, a five-carbon sugar, and a phosphate group. Nucleotides are essential in various biological processes, including DNA replication, transcription, and translation.
Oligonucleotide: An oligonucleotide is a short, synthetic polymer of nucleotides, typically ranging from 2 to 50 nucleotides in length. These molecules are essential in the context of DNA synthesis, as they serve as building blocks for the construction of longer DNA sequences.
Oligonucleotides: Oligonucleotides are short strands of nucleotides, typically made up of 20 or fewer bases, which are the building blocks of DNA and RNA. They are synthesized artificially to match specific sequences within a gene for purposes such as DNA synthesis, regulation, and analysis.
Phosphate Triester: A phosphate triester is a type of organic compound consisting of a phosphate group esterified with three alkyl or aryl groups. These compounds are important in the context of DNA synthesis, as they are involved in the formation of the phosphodiester backbone of DNA molecules.
Phosphite: Phosphite is an ion or salt containing the phosphite ion (PO3^3-), often involved in various chemical reactions including those in organic chemistry and biochemistry. In the context of DNA synthesis, it can be part of a reagent used for introducing phosphorus atoms into molecules.
Phosphite Triester: A phosphite triester is a type of organic compound that consists of a central phosphorus atom bonded to three organic substituents, typically alkyl or aryl groups. These compounds are important intermediates in the synthesis of DNA and other biomolecules.
Phosphodiester Bond: A phosphodiester bond is a covalent chemical bond that links the phosphate group of one nucleotide to the sugar (deoxyribose) of the next nucleotide, forming the backbone of DNA molecules. This bond is crucial for the structural integrity and information storage capabilities of DNA.
Phosphoramidite: Phosphoramidite is a key chemical building block used in the automated synthesis of DNA and RNA molecules. It is a reactive phosphorus-containing compound that is used to sequentially attach nucleotides to a growing oligonucleotide chain during solid-phase synthesis, allowing for the controlled, stepwise construction of synthetic genetic material.
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.
Pyridine: Pyridine is a heterocyclic aromatic organic compound with the chemical formula C₅H₅N. It is a colorless, volatile liquid with a distinctive unpleasant odor, and it is widely used in the production of various chemicals and pharmaceuticals.
Tetrazole: A tetrazole is a heterocyclic organic compound containing a five-membered ring composed of four nitrogen atoms and one carbon atom. It is an important functional group in organic chemistry with applications in medicinal chemistry, materials science, and coordination chemistry.
Trichloroacetic Acid: Trichloroacetic acid (TCA) is a colorless, crystalline organic compound with the chemical formula CCl3COOH. It is a strong acid commonly used in various applications, including as a reagent in biochemical and analytical procedures, particularly in the context of DNA synthesis.
Watson-Crick Base Pairing: Watson-Crick base pairing is the fundamental principle that governs the structure and replication of DNA. It describes the specific, complementary interactions between the nitrogenous bases that make up the DNA double helix.