Module 6

• 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


  (A)
• Nucleic acid purification


  1. Organic purification - phenol and chloroform/isoamyl alcohol (25:24:1) are mixed with an equal volume of samples by vortexing

  2. Inorganic purification - involves the incubation of nuclei with only proteinase K at 65°C

  
• DNA Spectrophotometry


  Determining nucleic acid purity and yield

  
• NanoDrop Spectophotometry


  Determining nucleic acid purity and yield

  
• Fluorometric Methods


  Determining nucleic acid purity and yield

  
• Real-Time PCR Method


  Determining nucleic acid purity and yield

  
• 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 a distance of migration of an unknown sample with DNA or RNA ladders either by visualization or by computer assisted measurement allows determination of nucleic acid size
  • DNA are reduced to fragments by digestion with restriction enzymes
  • RNA are small molecules and no digestion is required prior to electrophoresis

  
• DGGE


  The principle is that 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 to through a porous gel matrix 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


  • Use 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 - sample and probe in solution
  • Solid support hybridization - occurs in biphasic environment, solid phase - sample, liquid phase - probe
• Southern Blot


  • Described by E.M. Southern in 1975
  • Detect specific DNA sequences
  • Electrophoretic separation, transferring to solid support nitrocellulose and hybridization
  • Detects gene mutations

  
• Northern Blot


  • Uses RNA sample
  • RNA is extracted digested, electrophoresed, blotted and probed
  • Not routinely use in clinical lab (RNA not stable)

  
• Western Blot


  • Used to identify proteins separated by PAGE
  • Confirmatory for HIV
  • Diagnosis of Lyme disease
• RFLP


  • 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
  • Restriction fragments are separated on gel and analyzed by southern blotting techniques
  • Also used for polymorphism detection assays
  • DNA maybe amplified by PCR then subjected to RFLP analysis

  
• Amplification methods


  • In vitro techniques for replication of a target molecule from a very low quantity to detectable levels
  • Amplified nucleic acids, whether an RNA or DNA are referred to as amplicons

  
• Amplification methods


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

  • Synthesis of target nucleic acid sequence
  • Reverse-Transcriptase PCR - if the nucleic acid of interest is RNA rather than DNA, conversion of RNA to DNA using reverse transcriptase
  • Multiplex PCR - uses numerous primers for different targets in same mixture
  • Real-Time PCR - target amplification and detection steps occur simultaneously, PCR product is detected using fluorescent dyes

  
• Probe amplification methods


  • Uses probes or pieces of DNA or RNA that are labelled with a detectable molecule, such as biotin, dyes, or radioactive isotopes. These probes bind complementary to the target
  • 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 are designed that 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
  • Employed to screen interferon-α-resistant hepatitis C and detect mutations in human genes

  
• Signal amplification methods


  • Uses a stimuli to generate a signal, where the signal is proportional to the amount of the target sequence present in the clinical specimen assays
  • Increase concentration of label molecules attached to nucleic acid increases sensitivity
  • The number of target molecules is not altered
  • Examples: Branched DNA (bDNA), Hybrid capture assay

  
• Other amplification techniques


  • Strand displacement amplification (SDA) - fully automated method, dsDNA is target of exponential amplification, amplifies target nucleic acid without use of thermocycler
  • Transcription Mediated Amplification (TMA) - isothermal assay, targets either DNA or RNA, RNA is the amplified product, detection of M. tuberculosis
  • NASBA - Nucleic acid Sequence-Based Amplification - similar to TMA, but only RNA is targeted for amplification, detection of HIV and CMV

  
• Microarray (DNA Chip Technology)

  • Uses a collection of spots attached to a solid support that is capable of quantifying 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
  • After hybridization, chips are scanned with high speed detectors and intensity of spot is quantitated
  • Since this is chip-based, this method is considerably expensive
• 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, between individual nucleotides, or phosphate backbone
• Causes of DNA degradation

  • Nucleases
  • Oxidation
  • Hydrolysis
  • Radiation
• DNA is very sensitive and can easily degrade in certain conditions
• Storage temperatures for DNA sample

  • Short-term storage (weeks) - 4°C in Tris-EDTA
  • Medium-term storage (months) - -80°C in Tris-EDTA
  • Long-term storage (years) under ethanol - -80°C as a precipitate
  • Long-terms storage (decades) - -164°C or dried
• 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, use of chemical inhibitors (Ca2+, EDTA, SDS, 2 mercaptoethanol), protein precipitation removes nucleases
  • Store purified DNA correctly