MICRO EXAM 2 CH11 Flashcards
What is nucleic acid hybridization?
The formation of hydrogen bonds between nucleotides of SS of RNA or DNA that are complementary to each other.
What is duplex formation?
Two complementary strands of nucleic acids binding together to form a DS structure.
What is the target in SS nucleic acid molecules?
Sequence that will be identified that is either immobilized or suspended in solution.
What is the probe in SS nucleic acid molecules?
Short ssRNA or ssDNA oligonucleotide that is labeled with a chemical or fluorescent molecule; used to detect the target.
What is probe selection?
Determines recognition to the target sequence and forms the duplex/hybrid with every complementary sequence.
What is blotting?
Technique that transfer DNA or RNA or proteins from gel to membrane.
Types of blots?
Northern, Southern and western blots.
What is southern blot and its target?
DNA immobilized on solid support.
Uses restricition enzymes.
What is northern blot and its target?
RNA (usually mRNA) immobilized on solid support.
Does not use restricition enzymes.
How is protein extract separated in a western plot?
SDS-Page
What is western plot and its target?
Proteins immobilized on solid support.
How is protein blotted onto membrane?
Elecetrophoresis
What does In situ hybridization detect?
Detects DNA/RNA (viruses ini tissue) directly in cells and tissue with labeled probes encased in paraffin (wax).
What does NAAT stand for?
Nucleic Acid Amplification Test
What does NAATs detect?
Amplifies the target microorganisms nucleic acid in a sample in a short amount of time.
What does PCR stand for?
Polymerase Chain Reaction
1st step in basic PCR cycle?
Denatures dsDNA
Types of PCR methods?
Real Time-PCR, Reverse Transcriptase-PCR, multiplex PCR, Nested PCR
2nd step in basic PCR cycle?
Annealing of primer
3rd step in basic PCR cycle?
Extension of primer
What is needed for PCR?
Template DNA
Oligonucleotide primers
Thermostable DNA pol
How do you prevent contamination?
Having separate areas for each step, use of UV for decontamination, PPE
How do you analyze PCR products?
Gel electrophoresis
What does RT-PCR detect?
PCR amplicons (copies of DNA) after each cycle that has a fluorescence tag
What is the purpose of the melting curve?
Determine results of verify purity of PCR products when 50% has been hybridized.
What does Reverse Transcriptase PCR detect?
Detects mRNA products.
What enzyme is used to convert mRNA to cDNA?
RNA-dependent DNA pol
What does multiplex PCR detect?
Simultaneously detects two or more targets in the same sample
What does nested PCR detect?
Amplifies first target region then amplifies another target region from the first amplification.
What does MALDI-TOF stand for?
Matrix-Assisted Laser Desorption-Ionization Time Of Flight
What does MALDI-TOF method use to detect microorganism?
Mass spectrometry
1st step of MALDI-TOF?
Small amount of colony is applied to metal target.
2nd step of MALDI-TOF?
Energy absorbing chemical. matrix solution is added.
3rd step of MALDI-TOF?
Metal target is loaded into the ionization chamber.
4th step of MALDI-TOF?
Matrix lattice is pulsed with a UV laser.
