MBD Lec 1: Introduction to Molecular Biology and Diagnostics
Molecular Diagnostics
- Clinical areas = infectious disease, genetics, tumor markers
- For reference laboratory, expanded to routine laboratory
Molecular Composition and Structure
- DNA = adenine, cytosine, guanine, thymine
- Base pairing = adenine = thymine, guanine ≡ cytosine
- Purines (adenine, guanine) have double rings
- Pyrimidines (cytosine, thymine) have single ring
- Helical double-stranded DNA, stable at pH 4-9 (denature beyond)
- Denaturation turns double-stranded into single-stranded
- Melting point = 50% double-stranded into single-stranded
- Stringy viscous solution in vitro, 3 meters length
- RNA = adenine, cytosine, guanine, uracil
- Base pairing = adenine = uracil, guanine ≡ cytosine
- Irregular structure, sensitive to alkaline hydrolysis
Code Writing
- Code, hereditary information from parent cell to daughter cell
- Single-stranded for replication, transcription, translation
- Gene, hereditary unit or nucleotide sequence (all information)
- Messenger RNA, carry protein message into cytoplasm (for tRNA)
- Transfer RNA, amino acid transport (for protein synthesis)
- Codon, 3 nucleotide sequence, encode specific amino acids
- There are 20 amino acids, 64 possible codons
- Genome, encodes 64 codons (21 amino acids, 3 stop codons)
- Replication, reproduction from parent to daughter cell
- Transcription, transfer information from DNA into RNA
- Translation, formation of proteins from mRNA template
- Amplification methods, techinique that increases target amount
- Fluorescence, emission at longer wavelength (excited at short)
- Oligonucleotide, short single-stranded nucleic acid
- Probe, nucleic acid used to identify hydribization target
- Polymerase chain reaction, amplification method in vitro
- Polymorphism, differences in DNA sequences (small percentage)
Methodology Classifications
- Detection = chemiluminescence, electrophoresis, hybridization
- Amplification, does not require direct techniques
Classification of Specific Procedures
- Direct nucleic acid testing
- Southern blot, endonuclease retriction to cleave (into nucleus)
- Detected by agarose gel electrophoresis, transferred to paper
- Detected by hybridization oligonucleotide probe
- Fluorescence in-situ hybridization
- Detect targets of interest in cytology or histology specimens
- Staining of DNA sequences by fluorescence-labelled DNA probes
- Denaturation, hybridization to single-stranded DNA probes
- Fluorescent-labelled DNA probes, fluorescence signal analysis
- Involve exposing chromosome to fluorescent-labelled probe
- Probe sequence bind to corresponding sequence (detectable)
- DNA fingerprinting or restriction fragment length polymorphism
- Specific variations in DNA sequence analysis
- Cuts long DNA into shorter fragments (to isolate polymorphism)
- Used to distinguished individuals, populations, gene location
- Amplified nucleic acid testing
- Polymerase chain reaction (most common), ligase chain reaction
- Strand displacement amplification, branched DNA
- Transcription-mediated amplification
- Transcription-based amplification system
- Loop-mediated isothermal amplification
- Nucleic acid sequence-based amplification
Polymerase Chain Reaction Cycle
- Components = DNA, DNA primers, nucleotides
- (1) Denaturation (94-98'C) = double-stranded to single-stranded
- (2) Annealing (50-68'C) = single-stranded paired with DNA primer
- (3) Extension (72'C) = depends on DNA polymerase to amplify
- Principle = amplify or multiply target (incorporating enzymes)
- Incorporated enzymes = DNA ligase, DNA and RNA polymerase
- Reverse transcriptase (RNA), alkaline phosphatase, cleavase
- Amplification depends on
- Target, thermocycling requirement, detection techniques
- Polymerase and ligase chain reaction (only uses thermocycling)
- Methods differ on
- Target, amplification, enzyme and thermocycling, detection
- Target is most commonly amplified (probe and signal can also)
- Thermal cycling (thermocycling), variations in temperature
- Increased temperature, unfolds DNA (double to single-stranded)
- Decreased temperature, reconnection or reannealing
- Amplification issues = amplicon (product), contamination
- Resolution = perform QC prior testing (ensure results in range)
Hybridization
- Pairing or annealing of 2 DNA strands
- Double-stranded hybrids detected using probe, identifies target
- Categories = solid phase, solution phase
- Environemntal factors
- (1) Temperature = ↑ temperature (melt), ↓ temperature (join)
- (2) pH = ↑ alkaline (separate), ↓ acidic (force together)
- (3) Salt concentration = found in hybridization buffer
- (4) Guanine to cytosine ratio = ↑ ratio (longer separation)
- Stringency, condition under the target is exposed to probe
- ↑ stringency = probe cannot bind to target
- ↓ stringency = probe binds to unrelated targets
Detection
- Depends in direct and amplified methods, labels probe or target
- Chemiluminescence, detected by luminometer (acridinium ester)
- Electrophoresis, movement in matrix by electrical field
- Enzyme, enzyme and substrate principle, combine with fluorescent
- Fluorescence, measured by fluorometer or polarization
- Radioactivity, measured by scintillation counter (decay)
How to Select Methodology
- TAT, cost of methodology, patient population, competency
- Laboratory equipment and space, task complexity, reporting
Clinical Applications
- Nucleic acid alterations
- Mutation, deletion (missing nucleotide), insertion (extra)
- Missense (substitution), nonsense (early termination)
- Single nucleotide polymorphism, most common (cystine to thymine)
- Clinical areas = infectious disease, paternity, genetic testing
- Pharmacogenetics, prevneting adverse drug reaction (mortality)
- Advantages
- (1) Sensitivity = amplification (readily detected)
- (2) Specificity = minimal false-positives by target of interest
- (3) TAT = faster in comparison with conventional methods
- (4) Application = infectious disease, tumor marker detection
- Disadvantages
- (1) Cost = more expensive (standardized would lower costs)
- (2) Personnel requirements = undergo training and seminars
- (3) Laboratory space requirements = dedicated space
 AMPLIFICATION METHOD AMPLIFIES USE OF THERMOCYCLING
Polymerase Chain Reaction Target amplification using DNA polymerase Yes
Ligase Chain Reaction Amplification of a short DNA probe using DNA ligase Yes
Transcription-mediated Amplification Target amplification using reverse transcriptase & RNA polymerase No
Strand Displacement Amplification Target amplification using DNA polymerase that continuously displaces No
strands of DNA containing the target sequence
Branched DNA Signal amplification using alkaline phosphatase No
Loop-mediated Isothermal Amplification Target amplification of multiple DNA sequences in a loop pattern using No
DNA polymerase
Nucleic Acid Sequence-based Amplification Target amplification using 3 enzymes No
Q Beta Replicase Probe amplification - The concentration of an RNA probe increases if the No
target is present