VMA 5114: Principles of Infectious Diseases - Molecular Diagnostic Techniques

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Questions and Answers

What is the primary purpose of molecular diagnostic techniques in infectious diseases?

To increase the sensitivity of direct examination

To reduce the dependency on culture (correct)

To develop new immunologic methods

To analyze protein detection

What is the direction of negatively charged molecules in an electrophoretic field?

Remains stationary

Towards the negative end

Towards the positive end (correct)

Moves randomly

What is the function of restriction enzymes in RFLP?

To hybridize DNA probes

To amplify DNA sequences

To detect protein expressions

To cut DNA at specific recognition nucleotide sequences (correct)

What type of end is produced by the restriction enzyme EcoRI?

Sticky end Signup and view all the answers

What determines the mobility of molecules in electrophoresis?

Size and structure of the molecule Signup and view all the answers

How many restriction enzymes have been studied in detail?

Over 3000 Signup and view all the answers

What is the purpose of hybridization in molecular diagnosis?

To detect specific nucleic acid sequences Signup and view all the answers

What is the type of electrophoresis used to detect rotavirus strains?

SDS-PAGE Signup and view all the answers

What is the purpose of the "probe" in hybridization?

To detect complementary sequences in samples Signup and view all the answers

What is the difference between Southern and Northern blotting?

Southern blotting analyzes DNA, while Northern blotting analyzes RNA. Signup and view all the answers

What is a common application of RFLP analysis?

Identifying individuals in forensic investigations Signup and view all the answers

What does the abbreviation PCR stand for?

Polymerase Chain Reaction Signup and view all the answers

What is a potential disadvantage of PCR?

It can produce false positive results Signup and view all the answers

What is the purpose of primers in PCR?

To provide a starting point for DNA synthesis Signup and view all the answers

Which of these is NOT a type of hybridization technique?

Western blot Signup and view all the answers

What is the main principle behind polymerase chain reaction (PCR)?

Amplifying DNA sequences using a heat-stable polymerase Signup and view all the answers

What is the main purpose of using thermostable polymerases in PCR?

To prevent the degradation of DNA at high temperatures. Signup and view all the answers

What is the key difference between standard PCR and real-time PCR?

Standard PCR provides qualitative results, while real-time PCR provides quantitative results. Signup and view all the answers

What is the role of the primer binding sites in PCR?

They determine the size of the PCR product. Signup and view all the answers

Which of the following polymerase enzymes has the longest half-life at 95°C?

Deep Vent Signup and view all the answers

Which of the following PCR variations allows for the detection of multiple target sequences in a single reaction?

Multiplex PCR Signup and view all the answers

What is the main advantage of using LAMP over traditional PCR?

LAMP does not require the use of a thermal cycler. Signup and view all the answers

What is the function of the probe or dye in real-time PCR?

To quantify the amount of target DNA present in the sample. Signup and view all the answers

What is the main purpose of the lag phase in a qPCR curve?

To reflect the amount of target DNA present in the sample. Signup and view all the answers

Study Notes

Molecular Diagnostic Techniques

Molecular diagnosis involves the use of DNA, RNA, and proteins to identify pathogenic agents
Advantages of molecular diagnosis: • Increased sensitivity and specificity • Reduced dependency on culture • Safe

Electrophoresis

Separates molecules in an electrophoretic field based on: • Size: smaller molecules move faster • Structure: supercoiled > linear > nicked circles (DNA)
Example: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) used to detect rotavirus strains

Restriction Fragment Length Polymorphism (RFLP)

Analyzes differences among homologous DNA sequences using restriction enzymes
Restriction enzymes: • Cut DNA at specific recognition nucleotide sequences • Sequence specific
Examples of restriction enzymes: • EcoRI: creates sticky ends • SmaI: creates blunt ends
Over 3000 restriction enzymes have been studied, with more than 600 available commercially
Applications of RFLP: • Forensic analysis • Parenthood identification

Hybridization

Process where a probe binds to a complementary single-stranded sequence
Probe: • A fragment of nucleic acid • Labeled using radioisotope, enzyme, or chemiluminescence • Detects complementary sequences in samples • High degree of specificity • Varies in size
Types of hybridization: • Dot blot • In situ • Southern • Northern • Microarray

Nucleic Acid Amplification

Target amplification: an enzyme-mediated process to synthesize copies of targeted nucleic acid
Examples of nucleic acid amplification techniques: • Polymerase Chain Reaction (PCR) • Isothermal amplification (e.g., LAMP)
PCR: • Highly sensitive • Risk of false positives
PCR products: • 2n (where n is the number of cycles) • Example: 30 cycles can produce 1,073,741,824 products

PCR Primers

Single-stranded DNA fragments complementary to sequences flanking the region to be amplified
Factors affecting primer selection: • Distance between primer binding sites determines product size • Annealing temperature • Specificity • Nucleotide composition
Types of primers: • Random • Specific

PCR Variations

Reverse-transcriptase PCR
Nested PCR
Multiplex PCR
Quantitative or real-time PCR (qPCR)
Others

Real-Time or Quantitative PCR (qPCR)

Uses a probe or dye to generate a fluorescent signal from the product
Signal is generated in real-time, allowing for quantification of starting material
Threshold fluorescence level
Threshold cycles for each sample

LAMP (Loop-Mediated Isothermal Amplification)

Pros: • No thermal cycler needed • Quick (1 hour) • Sensitivity ≥ PCR • Visible results
Cons: • Design of primer sets can be complicated

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Description

This quiz covers molecular diagnostic techniques in infectious diseases, including electrophoresis, RFLP, hybridization, nucleic acid amplification, and protein detection. Diagnostic techniques such as direct examination, culture, immunologic methods, and molecular analysis are also discussed.