Molbio

• DNA purity and yield determination

  Reliable methods are crucial for various molecular applications
• Methods for DNA concentration determination

  • Each method has its own pros and cons
  • The choice depends on user preference and convenience
• UV spectrophotometry


  1. Placing the sample into a quartz cuvette

  2. Passing UV light through the sample at a specified path length

  3. Measuring absorbance at 260 nm (A260) for nucleic acid and A280 for contaminating protein
• Nucleic acids


  Have specific absorption peaks at 260 nm due to conjugated double bonds in their purine and pyrimidine rings
• A260/A230 ratio


  Another measure of purity, with a ratio above 1.4 typically desired for multiplex PCR methods
• Conversion factor from optical density to concentration

  1 unit at 260 nm = 50 mg/L of double-stranded DNA, 40 mg/L of RNA
• UV spectrophotometry

  • Simple, doesn't require large sample volumes, additional reagents, or incubation time
  • Drawbacks: Minimum sample volume requirement, inability to distinguish between DNA/RNA or double-stranded/single-stranded DNA, influence of biological contaminants, pH changes affecting UV readings, standardization of methods and buffers crucial for accurate quantitation
• NanoDrop spectrometry


  An extension and improvement of UV spectrophotometer, combining fiber optic technology and surface tension properties to capture and retain small sample volumes (1-2 μL)
• NanoDrop spectrometry


  • Requires minimal sample volume, facilitating additional quality control steps
  • Displays entire absorbance spectrum of the sample in graphical form
  • Facilitates detection and identification of contaminants
  • Can determine a wide range of sample concentrations without requiring serial dilutions
  • Reduces the need for sample cleanup and salt removal
  • Improves downstream molecular analysis, particularly in PCR reactions
• Fluorometric methods


  Highly sensitive nucleic acid quantitation methods, involving dyes intercalating and binding to nucleic acid grooves nonspecifically or selectively
• Commonly used fluorometric dyes

  • Ethidium bromide
  • Hoechst 33258
  • PicoGreen
• Fluorometric methods


  • Drawbacks: Need for costly equipment, expensive proprietary reagent kits, lengthy assay setup and dye incubation time, sample volume consumption, accuracy affected by DNA fragmentation and presence of contaminants
  • Benefits over UV spectroscopy: Low sample volume requirement, high sensitivity allows detection and quantitation of very small sample concentrations, maximum sample preservation for downstream applications
• Real-time PCR (qPCR)


  • Advantages: Ability to assess the amount of target DNA, detection of PCR inhibitors, specificity inherent in the assay using fluorometric probes
  • Drawbacks: Expensive proprietary reagents, primers, and probes, specialized and costly instrumentation, lengthy assay time, measurement accuracy may depend on qPCR assay design and DNA fragmentation, sample volume expended for concentration determination
• Gel electrophoresis


  Routine method for detection and size analysis of proteins and nucleic acids, where charged biomolecules are propelled through a porous gel matrix by an electrical current
• Gel electrophoresis


  • Protein analysis often performed using vertically oriented polyacrylamide gels, DNA and RNA analysis commonly conducted using horizontal agarose slab gels
  • DNA and RNA are negatively charged due to their phosphore-sugar backbone, migrating towards the positive pole in the electric field
  • Migration rate depends on molecule shape and charge-to-mass ratio
  • Separation matrices include agar, agarose, polyacrylamide, and composite agarose-acrylamide gels, providing a tortuous path for DNA migration and enabling separation by molecular mass
• Agarose gel electrophoresis


  Agarose is a purified linear galactan hydrocolloid from marine algae, forming a linear alternating copolymer of D-galactose and 3,6-anhydro-L-galactose, which dissolves in aqueous solution and forms a gel upon cooling
• Agarose gel matrix


  • Negatively charged due to anionic groups affixed to the matrix, with dissociable cations migrating toward the cathode and causing electroosmotic flow
  • Orientation of agarose gel fibers and fiber bundles affects electrophoresis, with preelectrophoresis in a direction perpendicular to the eventual direction of electrophoresis creating skewed lanes that gradually straighten out
• Size of DNA molecules and mobility in agarose gels

  Mobility influenced by relative size to average pore size of gel matrix, with mobility decreasing linearly with increasing molecular mass in low-voltage electric fields, and becoming nearly independent of molecular mass at higher electric fields or for DNA molecules larger than 12 kbp
• Chromosome


  Carries genes for expression, replicates DNA sequences in the cell cycle
• The faithful transmission of genetic information from one generation to the next depends on a cell's ability to make copies of each chromosome and then distribute the complete set of chromosomes to the two daughter cells
• Chromosomes


  • Have many features to ensure their proper duplication and distribution during the cell-division events
• Cell Cycle


  A highly coordinated sequence of events that occurs during the division process
• Phases of the cell cycle

  • G1 phase (Gap phase 1)
  • S phase
  • G2 phase
  • M phase
• G1 phase


  Cell growth occurs until cells attain a minimum size that is required to progress to the next phase
• G2 phase


  The cell prepares for mitosis
• S phase


  The DNA is replicated, thereby duplicating all of the chromosomes. The two identical copies of each chromosome are called sister chromatids, and they remain physically associated with one another
• M phase


  Sister chromatids are separated and a complete set of chromosomes is delivered to separate pole of the cell. This process is known as chromosome segregation and is followed by Cytokinesis, which completes the division of the mother cell into two daughter cells, each containing the same number of chromosomes
• Interphase


  G1, S, and G2 phases
• Mitotic phase


  M phase
• During interphase, chromosomes are replicated. During mitosis they become highly condensed and then are separated and distributed to the two daughter nuclei
• Mitotic chromosomes


  The highly condensed chromosomes in a dividing cell
• Chromosome dynamics during cell cycle

  1. Nuclear membrane disassembly

  2. Sister chromatid condensation

  3. Microtubule binding to centromeres

  4. Kinetochore formation

  5. Sister chromatid separation

  6. Nuclear membrane reformation
• Karyotype


  The display of the chromosome set of an individual, lined up from the largest to the smallest
• Genome


  The total DNA content of the cell, divided among one or more chromosomes
• Chromosome organize, store, and transmit genetic information and they must be compacted significantly to fit within cells
• Nucleoid


  The compact bacterial chromosome
• Bacterial genome


  • Organized into definite bodies, occupying about a third of the cell volume
  • Contains many independent chromosomal domains, each a supercoil loop of DNA
  • Majority are circular, some are linear
• Organization and dynamics of bacterial chromosome

  1. Pre-replication nucleoid is ellipsoidal and helical

  2. DNA organized into parallel bundles that rotate

  3. Nucleoid density fluctuates cyclically

  4. Nucleoid length varies discontinuously, in a cyclic pattern
• Nucleoid-associated proteins (NAPs)


  DNA-binding proteins that influence global chromosome organization and transcriptional patterns
• NAPs


  • H-NS, HU, IHF, FIS