MolBio

• Nucleic acid

  Macromolecules that exist as polymers called polynucleotides
• Nucleic acid structural unit

  • Composed of three (3) essential components
• Base pairing of nucleic acid forms the basis for DNA replication, RNA transcription and translation into protein

  
• Enzymes modify nucleic acids (polymerases, transcriptases, ligases, and nucleases)
• Formation of nucleic acid


  Shown in diagram (A)
• Organic purification


  Phenol and chloroform/isoamyl alcohol (25:24:1) are mixed with an equal volume of samples by vortexing
• Inorganic purification


  Involves the incubation of nuclei with only proteinase K at 65°C

  
• Methods for determining nucleic acid purity and yield

  • DNA Spectrophotometry
  • NanoDrop Spectophotometry
  • Fluorometric Methods
  • Real-Time PCR Method

  
• Nucleic acid analyses


  • Electrophoresis
  • Hybridization Assay
  • Amplification Techniques
  • DNA Sequencing
  • Polymorphism Based
• Electrophoresis


  • Movement of DNA or RNA in response to an electric field
  • Comparison of migration distance of unknown sample with DNA or RNA ladders allows determination of nucleic acid size

  
• DGGE (Denaturing Gradient Gel Electrophoresis)


  A linearly increasing gradient of denaturants is added to the polyacrylamide gels so that DNA fragments of the same length but with different base-pair sequences can be separated

  
• Molecular separation of DNA

  • Routinely uses gel electrophoresis
  • Uses electrical current to propel charged molecules through a porous gel matrix at a rate that is a function of the charge, size, and shape of the molecules
  • Uses either TAE or TBE
• Nucleic acid hybridization


  • The interaction between single stranded nucleic acid to form a (duplex) double stranded molecules based on complementary base pairing of the sequences
  • Binding between strands is both reversible and base-specific

  
• Annealing


  Process of recombination of two non-labeled strands into a stable double stranded structure
• Hybridization


  • If one strand has a marker, a hybrid is formed between labeled and an unlabeled strand
  • The labeled strand is called the probe
  • The product is a hybrid
• Primers


  • Short nucleotide sequence complementary to a specific DNA sequence and initiate DNA replication
  • Provides a free 3'-OH end for DNA polymerase to start synthesis of chain

  
• Probes


  • Used to target a particular sequence of complementary DNA or RNA
  • Can be labeled with radionuclide (P32), enzyme, and biotin

  
• Hybridization assay formats


  • Liquid or Solution Phase Hybridization
  • Solid Support Hybridization
• Molecular blotting techniques


  • Southern Blot: Detect specific DNA sequences
  • Northern Blot: Uses RNA sample
  • Western Blot: Used to identify proteins separated by PAGE

  
• RFLP (Restriction Fragment Length Polymorphism)


  • Results from a variable number of tandem repeats (VNTR) in a short DNA fragment
  • Used in forensic diagnosis, donor transplantation, identify carriers of mutated genes

  
• Amplification methods


  • Target amplification
  • Probe amplification
  • Signal amplification
• Target amplification method - PCR

  • Synthesis of target nucleic acid sequence
  • Modifications: Reverse-Transcriptase PCR, Multiplex PCR, Real-Time PCR

  
• Probe amplification methods


  • Uses probes or pieces of DNA or RNA that are labelled with a detectable molecule
  • Examples: ligase chain reaction, cycling probe technology, cleavase invader technology

  
• Cleavase/Invader technology


  • Relies on specific recognition and cleavage of particular DNA structures by flap endonuclease-I family of DNA polymerases
  • Two primers hybridize target sequence in an overlapping manner
  • Signal and target probes bind to target sequence
  • Enzyme cleavage of probe-test sample hybrid will yield fluorescent signal

  
• Signal amplification methods


  • Uses a stimuli to generate a signal, where the signal is proportional to the amount of the target sequence present
  • Increase concentration of label molecules attached to nucleic acid increases sensitivity
• Other amplification techniques


  • Strand Displacement Amplification (SDA)
  • Transcription Mediated Amplification (TMA)
  • Nucleic Acid Sequence-Based Amplification (NASBA)
• Microarray (DNA Chip Technology)

  • Uses a collection of spots attached to a solid support that is capable of quantitating hundreds or even thousands of genes in a cell or a tissue, simultaneously
  • Chips are used to examine activity of gene fragments using hybridization reaction between the microarray and fluorescent sample
• Denaturation


  • Separation of dsDNA to ssDNA
  • Breaking of H bonds between base pairs
• Degradation


  Breaks in the backbone of DNA molecule either dsDNA or ssDNA
• Causes of DNA degradation


  • Shown in diagram
• Short-term DNA storage


  4°C in Tris-EDTA (weeks)
• Medium-term DNA storage


  • 80°C in Tris-EDTA (months)
• Long-term DNA storage


  • -80°C as a precipitate under ethanol (years)
  • -164°C or dried (decades)
• Steps to prevent DNA degradation


  • Correct handling and storage of materials
  • Perform extractions at 4°C, on ice or in the cold
  • Inhibit nuclease activity (low temperature, chemical inhibitors, protein precipitation)
  • Store purified DNA correctly