lecture 4 Flashcards
What are the main steps in nucleic acid extraction?
The main steps are cell lysis, separation of nucleic acids from other cellular components, and purification of the nucleic acids.
What are the advantages of molecular tests over conventional methods?
High sensitivity and specificity, rapidity, high throughput, applicability to archival material, drug targeting of specific mutations/proteins, and better understanding of resistance mechanisms.
What are the limitations of molecular tests?
Inability to distinguish live from dead pathogens, high costs, high technical skill requirement, limited availability, and lack of national and international standardization of assays.
What are the major steps in PCR?
Denaturation, annealing, and elongation.
What is the purpose of using EDTA in whole blood or plasma samples?
EDTA is used as an anticoagulant to prevent clotting, and it is preferred over heparin because heparin inhibits PCR.
What are the key components required for PCR?
A thermostable DNA polymerase, deoxynucleotides (dNTPs), the target sequence, and a pair of oligonucleotide primers.
What is the significance of dried blood spots (DBS) in molecular diagnostics?
DBS are used to reliably obtain nucleic acid from whole blood, and they must be thoroughly air-dried and stored in a desiccant-containing sealed bag to prevent moisture accumulation and microbial growth.
What are the three main stages of PCR?
Denaturation (94-98°C), annealing (50-65°C), and elongation (72°C).
What are the common specimen types used in molecular diagnostics?
Whole blood, serum, plasma, urine, feces, saliva, sputum, spinal fluid, synovial fluid, amniotic fluid, pleural fluid, pericardial fluid, gastric fluid, ascitic fluids, swabs, and various tissues.
What are the main reasons for rejecting a sample in molecular diagnostics?
Unreadable information on the sample tube, mismatched sample and form labeling, samples without request forms, insufficient sample volume, and hemolyzed samples.
What are the key differences between solid-phase and solution-based nucleic acid extraction methods?
Solid-phase methods are easier to operate, allow for large batch processing, are free from toxic chemicals, and are highly reproducible. Solution-based methods are favored for large quantities of DNA or large sample volumes but have limited throughput due to manual steps.
What is the role of chaotropic salts in solid-phase nucleic acid extraction?
Chaotropic salts, such as guanidine thiocyanate, help in the adsorption of DNA onto silica-coated magnetic particles.
What is the purpose of the annealing step in PCR?
The annealing step allows the primers to bind to the complementary sequences on the single-stranded DNA template.
What are some other amplification methods besides PCR?
Single molecule PCR/Digital PCR, asymmetrical and allele-specific PCR, ligase chain reaction, transcription-based amplification method, and strand displacement amplification (SDA).
What are the key factors that affect PCR efficiency?
The concentration of primers and DNA polymerase, the temperature cycling protocol, and the presence or absence of polymerase inhibitors.
What is the importance of proper specimen handling and preservation in molecular diagnostics?
Proper labeling, preservation, and timely separation of specimens ensure accurate diagnosis and prevent contamination or degradation of nucleic acids.
What are the indications for molecular testing?
Monitoring viral load, rapid diagnosis of viral infections (e.g., HIV in infants), providing a prognosis based on mutations, paternity testing, and tracking outbreaks of infections.
What are the key components of a quality system in molecular diagnostics?
Monitoring the entire system, detecting and limiting errors, and improving consistency among different testing sites.
What is the role of thermal stable polymerases in PCR?
Thermal stable polymerases, such as Taq polymerase, allow PCR to be performed at high temperatures without denaturing the enzyme, enabling repeated cycles of DNA amplification.
What is the significance of the elongation step in PCR?
During elongation, the DNA polymerase extends the primers by adding nucleotides to synthesize a new strand of DNA complementary to the template strand.
