Chapter 11: Molecular Techniques Flashcards
What represents a significant advancement in clinical microbiology?
Molecular Diagnostics
Considered the fastest growing section in many clinical laboratories.
What do molecular diagnostics provide to clinicians?
Rapid answers for treatment options
Saves valuable time in cases of life-threatening infections.
When are molecular techniques particularly useful?
When traditional methods are not practical or available.
Which organisms cannot be routinely cultivated?
Viruses and Chlamydia
What is required for the growth of Mycobacterium tuberculosis?
A long incubation period
What are genetic probes?
Short, specific sequences of single-stranded DNA or RNA
What do genetic probes identify within nucleic acid?
One or more sequences of interest
What does the degree of hybridization depend on?
Base sequence homology and reaction conditions
What are the conditions that affect stringency?
Length of probe, probe concentration, degree of complementarity, salt concentration, temperature, pH, concentration of destabilizing agents
What determines the detection of hybridization?
Type of label
What is the most common method for detection in clinical microbiology?
Chemiluminescence
List the formats of nucleic acid probes.
- Liquid phase
- Solid phase
- Southern blot
- Northern blot
- In situ hybridization
What is signal amplification in nucleic acid amplification methods?
Increased concentration of labeled molecule without increasing probe or target concentration.
Branched DNA amplification:
Solid phase sandwich hybridization that uses multiple sets of synthetic probes
Hybris capture assays:
Solution hybridization antibody capture
Target amplification:
– Enzymes produce copies of target nucleic acid which are detected by primers that bind to complementary strands.
– Produces millions of copies of targeted sequence which act as templates for subsequent amplification cycles.
– Polymerase chain reaction (PCR)
What is the purpose of Polymerase Chain Reaction (PCR)?
To produce millions of copies of targeted nucleic acid sequence
What is the first step in the Polymerase Chain Reaction?
Denature nucleic acid into single stranded DNA
What is Reverse Transcriptase PCR (RT-PCR) used for?
To detect RNA viruses
What does Nucleic Acid Sequence-Based Amplification (NASBA) require?
Three enzymes: AMV reverse transcriptase, Ribonuclease H, T7 RNA polymerase
Target could be DNA or RNA most often RNA viruses
What is Transcription-Mediated Amplification (TMA) developed for?
Detection of C. trachomatis and N. gonorrhoeae
Targets rRNA
What is strain typing and identification based on?
DNA or genetic fingerprinting and genetic relatedness
What are Amplification Independent Methods?
- Restriction Fragment Length Polymorphisms (RFLPs)
- Southern blotting
- Plasmid profile analysis
- Multilocus enzyme electrophoresis (MLEE)
List some Amplification Dependent Methods.
- Random amplified polymorphic DNA
- Repetitive palindromic extragenic elements PCR
- Multilocus sequence typing
What is a DNA microarray?
Grouping at the micron level of DNA molecules attached to a solid support
What are DNA chips used for?
Investigation of gene expression from an entire organism
What is the purpose of nanoarrays?
To analyze proteins and gene expression at nanometer spatial resolutions
What is Sanger Sequencing?
Automated DNA sequencing method
What does MALDI-TOF stand for?
Matrix-Assisted Laser Desorption–Ionization Time of Flight
What is the primary use of MALDI-TOF?
Rapid identification of microorganisms
What are the three basic nucleic acid molecular diagnostics applications?
- Nucleic acid hybridization assays
- Nucleic acid amplification techniques
- Strain typing techniques
Nucleic acid hybridization assays – use
labeled probes
Nucleic acid amplification techniques – use
PCR and varients
