🔮Chemical Basis of Bioengineering I Unit 13 – Nucleic Acids & Molecular Genetics

Key Concepts

• Nucleic acids (DNA and RNA) store, transmit, and express genetic information in living organisms
• DNA consists of four nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
  • Bases pair through hydrogen bonding: A with T and G with C
• RNA consists of four nucleotide bases: adenine (A), uracil (U), guanine (G), and cytosine (C)
  • Bases pair through hydrogen bonding: A with U and G with C
• Central dogma of molecular biology describes the flow of genetic information: DNA → RNA → protein
• Genetic code is the set of rules that translates nucleotide sequences into amino acid sequences
  • Codons are three-nucleotide sequences that specify amino acids or stop signals
• Gene expression involves transcription of DNA into RNA and translation of RNA into proteins

Structure and Function

• DNA is a double-stranded helix composed of nucleotides connected by phosphodiester bonds
  • Nucleotides consist of a sugar (deoxyribose), a phosphate group, and a nitrogenous base
• Complementary base pairing and hydrogen bonding stabilize the DNA double helix
• RNA is typically single-stranded and composed of nucleotides connected by phosphodiester bonds
  • Nucleotides consist of a sugar (ribose), a phosphate group, and a nitrogenous base
• RNA can form secondary structures (hairpins, loops) through intramolecular base pairing
• DNA stores genetic information, while RNA plays roles in gene expression and regulation
  • Messenger RNA (mRNA) carries genetic information from DNA to ribosomes for protein synthesis
  • Transfer RNA (tRNA) delivers amino acids to ribosomes during protein synthesis
  • Ribosomal RNA (rRNA) is a component of ribosomes and catalyzes peptide bond formation

DNA Replication

• DNA replication is the process of copying DNA molecules during cell division
• Replication is semiconservative: each daughter DNA molecule contains one original and one newly synthesized strand
• Replication begins at specific sites called origins of replication
  • Helicase unwinds the DNA double helix, separating the strands
  • Single-stranded binding proteins stabilize the separated strands
• DNA polymerase synthesizes new DNA strands using the original strands as templates
  • Leading strand is synthesized continuously in the 5' to 3' direction
  • Lagging strand is synthesized discontinuously as Okazaki fragments
• DNA ligase joins Okazaki fragments to form a continuous strand
• Proofreading and repair mechanisms ensure high fidelity of DNA replication

Transcription and Translation

• Transcription is the synthesis of RNA from a DNA template
  • RNA polymerase binds to a promoter region and separates the DNA strands
  • RNA polymerase synthesizes a complementary RNA strand in the 5' to 3' direction
  • Transcription ends at a terminator sequence, releasing the RNA transcript
• Eukaryotic mRNA undergoes post-transcriptional modifications (5' capping, 3' polyadenylation, splicing)
• Translation is the synthesis of proteins using mRNA as a template
  • Ribosomes, composed of rRNA and proteins, catalyze protein synthesis
  • tRNAs deliver amino acids to the ribosome based on codon-anticodon recognition
  • Ribosomes synthesize proteins by forming peptide bonds between amino acids
• Genetic code is degenerate: multiple codons can specify the same amino acid
  • Start codon (AUG) initiates translation, and stop codons (UAA, UAG, UGA) terminate translation

Gene Regulation

• Gene regulation controls the timing, location, and amount of gene expression
• Prokaryotic gene regulation often involves operons (lac operon, trp operon)
  • Repressors and activators bind to operator sequences to control transcription
• Eukaryotic gene regulation occurs at multiple levels: transcriptional, post-transcriptional, translational, and post-translational
  • Transcription factors bind to enhancer and silencer sequences to modulate transcription
  • Chromatin structure (histone modifications, DNA methylation) affects gene accessibility
• Alternative splicing generates multiple protein isoforms from a single gene
• RNA interference (RNAi) and microRNAs (miRNAs) regulate gene expression post-transcriptionally
  • Small interfering RNAs (siRNAs) and miRNAs guide the degradation or translational repression of target mRNAs

Mutations and DNA Repair

• Mutations are changes in the DNA sequence that can alter gene function
  • Point mutations involve single nucleotide changes (substitutions, insertions, deletions)
  • Frameshift mutations alter the reading frame, often resulting in nonfunctional proteins
• Mutations can be spontaneous or induced by mutagens (UV radiation, chemicals)
• DNA repair mechanisms maintain genomic integrity by correcting DNA damage
  • Base excision repair removes and replaces damaged bases
  • Nucleotide excision repair removes bulky DNA lesions
  • Mismatch repair corrects base-pairing errors during replication
• Defects in DNA repair pathways can lead to increased mutation rates and genetic disorders

Molecular Techniques

• Polymerase chain reaction (PCR) amplifies specific DNA sequences using primers and DNA polymerase
  • Denaturation, annealing, and extension steps are repeated to exponentially increase the target sequence
• DNA sequencing determines the nucleotide sequence of DNA fragments
  • Sanger sequencing uses dideoxynucleotides to terminate DNA synthesis at specific bases
  • Next-generation sequencing (NGS) technologies enable high-throughput, parallel sequencing
• Restriction enzymes cleave DNA at specific recognition sequences, generating fragments for analysis or cloning
• DNA cloning involves inserting a DNA fragment into a vector (plasmid, viral vector) and propagating it in a host cell
• Gel electrophoresis separates DNA or RNA fragments based on size and charge
  • Agarose gel electrophoresis is used for larger fragments, while polyacrylamide gel electrophoresis (PAGE) is used for smaller fragments

Applications in Bioengineering

• Recombinant DNA technology enables the production of valuable proteins (insulin, growth factors) in engineered cells
  • Genes of interest are inserted into expression vectors and introduced into host cells (bacteria, yeast, mammalian cells)
• Genetically modified organisms (GMOs) have been engineered for enhanced traits (pest resistance, nutrient content)
  • Transgenic plants and animals express foreign genes introduced through genetic engineering
• Gene therapy aims to treat genetic disorders by introducing functional copies of defective genes into cells
  • Viral vectors (adenoviruses, retroviruses) are commonly used to deliver therapeutic genes
• CRISPR-Cas9 is a powerful genome editing tool that enables precise modification of DNA sequences
  • Guide RNAs direct Cas9 nuclease to specific genomic locations for targeted cleavage and editing
• Synthetic biology applies engineering principles to design and construct novel biological systems
  • Synthetic gene circuits, metabolic pathways, and minimal genomes are being developed for various applications