DNA Structure, Function and Manipulation Notes

• Structure: DNA (deoxyribonucleic acid) is a complex double-stranded molecule that contains the genetic blueprint for all living organisms. Its structure resembles a twisted ladder, fundamentally known as the double helix, comprising two long strands of nucleotides that run in opposite directions. The sequence of these nucleotides encodes genetic information essential for the functioning of cells, organisms, and the continuity of life.

• Prokaryotes vs. Eukaryotes:

  • Prokaryotic cells (e.g., bacteria) have a single, circular chromosome located in a region known as the nucleoid. This simplicity allows for rapid reproduction and adaptability to diverse environments. Additionally, prokaryotes may contain small, extrachromosomal DNA known as plasmids, which can confer advantageous traits such as antibiotic resistance.

  • Eukaryotic cells (e.g., humans) contain linear DNA organized into multiple chromosomes, with 46 chromosomes present in human somatic cells. Unlike prokaryotes, eukaryotic DNA is tightly associated with histones, proteins that help package the DNA into structures called chromatin, facilitating regulation and access to genetic information.

Discovery of DNA Structure

• Key Scientists:

  • Chargaff (1950): He discovered base pairing rules, revealing that Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G). This foundational knowledge established the importance of base pairing in DNA replication and transcription.

  • Rosalind Franklin (1952): Her pioneering work with X-ray diffraction led to the confirmation of DNA’s double helix structure. Franklin's photographic evidence, known as Photo 51, provided critical insights into the helical nature of DNA.

  • Watson and Crick (1953): Building on Franklin’s work, they proposed the accurate full structure of DNA, which illuminated how genetic information is stored and inherited.

Chargaff’s Base Pairing Rules

• Percentage of bases found in various organisms when analyzed:

  • Virus Phage T2: A: 33%, T: 17%, G: 20%, C: 30%

  • E. coli: A: 24%, T: 16%, G: 30%, C: 30%

  • Human: A: 20%, T: 20%, G: 30%, C: 30%
    This data supports the idea that the ratios of base pairs are consistent across species, emphasizing a universal genetic code.

Composition of DNA

• Nucleotides: The building blocks of DNA, nucleotides consist of three components:

  • Phosphate group: Provides the backbone structure, linking nucleotides together through phosphodiester bonds.

  • Deoxyribose sugar: A five-carbon sugar that complements the phosphate group, forming the sugar-phosphate backbone.

  • Nitrogenous base: There are four bases: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). The sequence of these bases encodes genetic information that directs cellular processes.

• Bonds: Nucleotides link through phosphodiester bonds, which connect the 3' hydroxyl group of one nucleotide to the 5' phosphate group of the next, forming a continuous sugar-phosphate backbone essential for the stability and integrity of DNA.

Base Pairing and Strands

• Base Pairs: - Adenine (A) pairs with Thymine (T) through 2 hydrogen bonds, while Cytosine (C) pairs with Guanine (G) via 3 hydrogen bonds. This specificity in base pairing ensures accurate replication and transcription of genetic information.

• Strand Orientation: The two strands of DNA are antiparallel, meaning one strand runs in the 5' to 3' direction, and the other runs from 3' to 5'. This antiparallel arrangement is crucial for the function of enzymes that replicate and transcribe DNA.

DNA Replication

• Purpose: DNA replication is essential for cell division, ensuring that each daughter cell receives an identical copy of the DNA.

• Mechanisms Proposed:

  • Conservative Model: Suggests that both parental strands remain together after replication.

  • Semiconservative Model: Each new DNA molecule consists of one parental strand and one newly synthesized strand. This model has been experimentally confirmed and is the basis for the modern understanding of DNA replication.

  • Dispersive Model: Proposes that parental and new DNA strands are interspersed in both daughter molecules.

DNA Replication in Bacteria

• Origin of Replication: Each bacterial chromosome has a unique site where replication begins, ensuring the timely duplication of genetic material. This site is recognized by initiator proteins that recruit the replication machinery.

• Bidirectional Synthesis: DNA replication in bacteria occurs bidirectionally, moving outward from the origin of replication, allowing for efficient copying of the circular genome.

DNA Replication in Eukaryotes

• Initiation: - Helicase unwinds the DNA double helix at replication origins, creating replication forks.

  • Topo-isomerase alleviates supercoiling ahead of the replication fork, preventing torsional strain.

  • Single-strand binding proteins (SSBs) stabilize and keep the unwound strands apart during replication.

  • Primase synthesizes short RNA primers, providing a starting point for DNA polymerization.

• Elongation: - DNA Polymerase III adds complementary DNA nucleotides to the RNA primer, leading to the production of a continuous leading strand (which requires only one primer) and a lagging strand (which is synthesized in segments known as Okazaki fragments, requiring multiple primers).

• Termination: Replication concludes when the replication forks meet, and DNA ligase joins any gaps between Okazaki fragments, ensuring that the newly formed DNA strands are continuous.

RNA Structure

• RNA (ribonucleic acid) is typically single-stranded and contains a ribose sugar backbone, differing from DNA's deoxyribose. RNA plays essential roles in coding, decoding, regulation, and expression of genes.

• The nitrogenous bases in RNA include Adenine (A), Uracil (U), Guanine (G), and Cytosine (C); Uracil replaces Thymine found in DNA, allowing for the unique functions of RNA.

Central Dogma of Molecular Biology

• The central dogma describes the flow of genetic information within a biological system, encompassing two main processes:

  • Transcription: The process in which a segment of DNA is copied into messenger RNA (mRNA) in the nucleus.

  • Translation: The mRNA is then translated into a polypeptide chain (protein) at the ribosome, where the specific sequence of amino acids is determined by the codons in the mRNA.

Transcription Process in Eukaryotes

• The transcription process occurs in the nucleus and involves several crucial steps:

  • Initiation: Transcription factors bind to the promoter region of a gene, facilitating the recruitment of RNA polymerase.

  • Elongation: RNA polymerase synthesizes the RNA strand in a 5' to 3' direction, matching the DNA template with complementary nucleotide bases.

  • Termination: The process concludes upon reaching a termination sequence; the pre-mRNA transcript is then modified by splicing, where introns (non-coding regions) are removed and exons (coding regions) are joined together to produce mature mRNA, ready for export to the cytoplasm.

Translation Process in Eukaryotes

• Occurs at the ribosome, involving mRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). The sequence of events in translation includes:

  • Initiation: The start codon (AUG) binds to the corresponding tRNA molecule, bringing together the ribosomal subunits.

  • Elongation: As the ribosome moves along the mRNA, tRNA molecules bring specific amino acids that are linked together, forming a polypeptide chain based on the sequence of codons found in the mRNA.

  • Termination: The process ends when a stop codon is reached, signaling the release of the completed polypeptide from the ribosome.

Gene Expression

• Gene expression is regulated by the structure of chromatin, where DNA wrapped around histone proteins forms nucleosomes.

  • Heterochromatin: Tightly wound and transcriptionally inactive, often found in regions of the genome that are not expressed.

  • Euchromatin: Loosely wound and accessible, contains active genes that can be expressed in response to physiological needs.

Restriction Enzymes Discovery

• Discovered in the 1950s-60s; these enzymes cut DNA at specific sequences, playing a critical role in the bacterial defense mechanism against viruses. They are essential tools in molecular biology and genetic engineering, enabling the manipulation of genetic material.

Applications of Restriction Enzymes

• Restriction enzymes are extensively utilized in genetic engineering for various applications, including DNA cloning, recombinant DNA creation, and genetic mapping. Ethical issues surrounding genetic manipulation raise concerns about unintended consequences and equitable access to genetic technologies, highlighting the need for thoughtful regulation.

Mapping Genomes

• The specific patterns generated by restriction enzymes are valuable for mapping gene locations within genomes and are fundamental in forensic analysis, enabling DNA profiling techniques that can identify individuals in legal contexts.

PCR (Polymerase Chain Reaction)

• PCR is a powerful technique used to amplify specific DNA segments through cycles of denaturation, annealing, and extension phases. This method has transformative applications in forensics, genetic testing, and research, allowing for the generation of millions of copies of a target DNA sequence from minimal starting material.

Recombinant DNA Technology

• This technology integrates genetic material from multiple sources, involving several key procedures: selecting an insert DNA fragment, choosing appropriate vectors for insertion, utilizing restriction enzymes for cutting and joining DNA segments, and introducing recombinant DNA into host cells for expression and propagation.

Genomics and Biotechnology

• Genomics focuses on the complete sets of genes within an organism, which is critical for advancements in gene therapy, personalized medicine, and understanding complex hereditary diseases. Techniques like CRISPR-Cas9 have revolutionized genetic editing capabilities, leading to potential applications in agriculture, medical therapies, and beyond.

Ethical Considerations in Genomics

• As biotechnology evolves, ethical considerations increasingly come into play, encompassing concerns about cloning, genetic modification impacts, accessibility of novel treatments, human enhancement, and cultural perspectives on genetic engineering practices.

Recent Techniques

• Recent advances, including base editing and organoid cultures, facilitate in-depth studies of diseases, providing insights that drive drug development and disease modeling for advancements in therapeutic strategies.