Molecular Diagnostic Techniques

Diagnostic Techniques

• Diagnostic techniques used to identify pathogenic agents include: direct examination, culture, immunologic methods, and molecular analysis.

Molecular Diagnosis

• Molecular diagnosis uses DNA, RNA, and proteins to identify pathogenic agents.
• Advantages of molecular diagnosis include: • High sensitivity (true positive rate) • High specificity (true negative rate) • Reduced dependency on culture • Safe

Molecular Diagnostic Tests

• Types of molecular diagnostic tests include: • Electrophoresis • Restriction fragment length polymorphism (RFLP) • Hybridization • Nuclear acid amplification (target) • Protein detection

Electrophoresis

• Electrophoresis separates molecules in an electrophoretic field.
• Negatively charged molecules move towards the positive end.
• Mobility of molecules depends on: • Size: smaller molecules move faster • Structure: supercoiled molecules move faster than linear molecules, which move faster than nicked circles (in the case of DNA)

Restriction Fragment Length Polymorphism (RFLP)

• RFLP involves analyzing differences among homologous DNA sequences through the use of restriction enzymes
• Restriction enzymes cut DNA at specific recognition nucleotide sequences, which are sequence-specific
• This technique is used to identify variations in DNA sequences by comparing the resulting fragment lengths

Hybridization

• Hybridization involves binding of a probe to complementary single-stranded DNA sequences.
• Denatured, single-stranded DNA is used in hybridization reactions.
• Examples of hybridization techniques include Dot, in situ, Southern, Northern, and microarray.

Probe Characteristics

• A probe is a fragment of nucleic acids used to detect complementary sequences in samples.
• Probes can be labeled using radioisotopes, enzymes, or chemiluminescence.
• Probes exhibit a high degree of specificity when binding to target sequences.
• Probes can vary in size.

Nucleic Acid Amplification

• Target amplification is an enzyme-mediated process that synthesizes copies of targeted nucleic acid
• Two types of nucleic acid amplification are: Polymerase Chain Reaction (PCR) and Isothermal amplification (e.g. LAMP)

Polymerase Chain Reaction (PCR)

• PCR is a highly sensitive method, but may produce false positives
• PCR Primers are single-stranded DNA fragments complementary to sequences flanking the region to be amplified
• The distance between the primer binding sites determines the size of the PCR product
• Primers determine the specificity of the PCR reaction

Features of Primers

• Types of primers: Random and Specific
• Primer characteristics: • Product size • Annealing temperature • Specificity • Nucleotide composition

PCR Variations

• Reverse-transcriptase PCR
• Nested PCR
• Multiplex PCR
• Quantitative or real-time PCR

Real-Time or Quantitative PCR (qPCR)

• qPCR uses a probe or dye to generate a fluorescent signal from the product.
• The fluorescent signal is measured in real-time, allowing for quantification of the starting material.

qPCR Signal Curve

• The qPCR signal curve is exponential in shape, consisting of three phases: lag phase, log phase, and stationary phase.
• The lag phase is inversely proportional to the amount of starting material.