Questions and Answers
What is the primary purpose of molecular diagnostic techniques in infectious diseases?
What is the direction of negatively charged molecules in an electrophoretic field?
What is the function of restriction enzymes in RFLP?
What type of end is produced by the restriction enzyme EcoRI?
Signup and view all the answers
What determines the mobility of molecules in electrophoresis?
Signup and view all the answers
How many restriction enzymes have been studied in detail?
Signup and view all the answers
What is the purpose of hybridization in molecular diagnosis?
Signup and view all the answers
What is the type of electrophoresis used to detect rotavirus strains?
Signup and view all the answers
What is the purpose of the "probe" in hybridization?
Signup and view all the answers
What is the difference between Southern and Northern blotting?
Signup and view all the answers
What is a common application of RFLP analysis?
Signup and view all the answers
What does the abbreviation PCR stand for?
Signup and view all the answers
What is a potential disadvantage of PCR?
Signup and view all the answers
What is the purpose of primers in PCR?
Signup and view all the answers
Which of these is NOT a type of hybridization technique?
Signup and view all the answers
What is the main principle behind polymerase chain reaction (PCR)?
Signup and view all the answers
What is the main purpose of using thermostable polymerases in PCR?
Signup and view all the answers
What is the key difference between standard PCR and real-time PCR?
Signup and view all the answers
What is the role of the primer binding sites in PCR?
Signup and view all the answers
Which of the following polymerase enzymes has the longest half-life at 95°C?
Signup and view all the answers
Which of the following PCR variations allows for the detection of multiple target sequences in a single reaction?
Signup and view all the answers
What is the main advantage of using LAMP over traditional PCR?
Signup and view all the answers
What is the function of the probe or dye in real-time PCR?
Signup and view all the answers
What is the main purpose of the lag phase in a qPCR curve?
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
Studying That Suits You
Use AI to generate personalized quizzes and flashcards to suit your learning preferences.
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.