NUCLEIC ACID AMPLIFICATION TECHNIQUES II Flashcards
- Number of target nucleic acid sequences in the sample is not changed compared to the target amplification procedure, such as __.
PROBE AMPLIFICATION
- PCR
THREE MAJOR PROCEDURES of PROBE AMPLIFICATION
- Ligase Chain Reaction
- Standard Displacement Amplification
- QB Replicase
▪ Manufactured and marketed by Abbott
- commercially available
Ligase Chain Reaction
▪ Manufactured and marketed by Beckton Dickinson
- commercially available
Standard Displacement Amplification
▪ Manufactured and marketed by Vysis
- commercially available.
QB Replicase
- A method for amplifying synthetic primers/probes complementary to target nucleic acid.
- Similar to PCR.
- The entire target sequence must be known in order to prepare __.
- LIGASE CHAIN REACTION (LCR)
- oligonucleotide primers
Less risk of carryover contamination resulting in a positive test in bDNA assay than in the __.
BRANCHED DNA AMPLIFICATION
PCR
- is currently available for the
qualitative and quantitative detection of _, _, _
- bDNA signal amplification assay
- Hep B, Hep C, and HIV 1
Developed by _
Used primarily for the detection of molecular characterization of _ in Genitourinary specimens
Also available for the detection of _ and _
HYBRID CAPTURE ASSAYS
- DigeneDiagnostics
- Human Papilloma Virus (HPV)
- Hepatitis B and Cytomegalovirus (CMV)
Sensitivity for HPV is _ copies of viral DNA
The__ has a unique structure that is recognized by antibodies bound to the surface of a microtiter well captures by it
HYBRID CAPTURE ASSAYS
- 1000
- DNA/RNA Hybrid
Captured hybrids are detected by binding of__ in a typical sandwich assay
The substrate for the alkaline
phosphatase is added, and
_ is measured
- alkaline phosphate conjugated anti-DNA/RNA hybrid antibodies
- chemiluminescence
Detects target nucleic acids by using a series of probes that bind to the target and overlap
- Target is __ primarily __
CLEAVAGE-BASED AMPLIFICATION
- DNA polymorphisms ( Factor V Leiden mutations)
–Enzyme isolated from bacteria that recognizes overlapping
sequences of DNA and makes a cut in the overlapping piece
Cleavase
target sequences are detected using a synthetic
probe consisting of sequences of DNA-RNA-DNA
- Used to detect genes associated with antimicrobial resistance in bacteria
CYCLING PROBE
- Methicillin resistance (mecA) in Staphylococcus aureus
o Vancomycin resistance (vanAand vanB) in
Enterococcus
METHODS OF PROBE AMPLIFICATION
- Branched DNA Amplification
- Hybrid Capture Assay
- Cleavage-Based Amplification
- Cycling Probe
- In LCR, ___________is used to ligate the adjacent primers together.
dna ligase
can be used to detect point-mutation in a target sequence.
◦The point-mutation that occurs in
__ of px with _ is one of the 1st applications for it.
LCR
- beta globulin
- sickle cell disease
It differs from most of the previous methods, it is an isothermal amplification process.
It is more similar to LCR than PCR
STRAND DISPLACEMENT
AMPLIFICATION (SDA)
There are two stages to SDA process:
Target Generation Phase
Exponential Probe or Target Amplification Phase
is denatured by heating to_
At each end of the target sequence, a primer and a probe bind close to each other.
The probes have a recognition sequence for a __
. TARGET GENERATION PHASE
- 95°C
- restriction enzyme
_ extends the primers, incorporating a modified nucleotide,_
Exonuclease- deficient DNA polymerase derived from E. coli
- 2′ deoxyadenosine 5′-O-(1-thiotriphosphate) (dATPaS)
When the restriction enzyme is added to the double stranded probe DNA, only one strand of the probe will be cut due to the __ introduced in the extension
reaction.
EXPONENTIAL PROBE/TARGET
AMPLIFICATION PHASE
- dATPaS
APPLICATION
Detection of , AND _
Addition of a _ to the reaction produces a fluorescent signal that corresponds to the amount of amplified target.
EXPONENTIAL PROBE/TARGET
AMPLIFICATION PHASE
M. tuberculosis, C. trachomatis, N. gonorrhoeae
- fluorogenic probe
Method for amplifying probes that have specificity for a target sequence.
Named for the major enzyme that is used to amplify probe sequences.
Qβ REPLICASE
RNA-dependent RNA polymerase from the bacteriophage Qβ.
Qβ REPLICASE
Target nucleic acid can be DNA (which must first be denatured) or RNA. The target nucleic acid is added to a well containing __ forming a sequence of
hybridization, capture, wash and release step.
For the amplification step, the probe-bound template is
mixed with IT , which replicates the probe molecules.
Qβ replicase
- reporter probes
–RNA molecules that have specificity for the target sequence and also contain a promoter sequence _ that is recognized by the Qβ replicase.
- are allowed to hybridize to the template.
This replication is very efficient with the generation of _ in less than _
Reporter probes
- (midivariant-1)
- 10^6–10^9 RNA molecules/probe
- 15 minutes
APPLICATION of Reporter probes
Used primarily to amplify nucleic associated w/ infectious organisms:
Mycobacteria
Chlamydia
HIV
CMV
No. of target sequences does not change
Large amounts of signal are bound to the target sequences.
Because the number of target sequences does not change, procedures are inherently better at quantitating the amount of target sequences present in the clinical sample.
- several are available commercially
SIGNAL AMPLIFICATION
SIGNAL AMPLIFICATION METHODS:
1
2
3
4
- Branched DNA Amplification
- Hybrid Capture Assay
- Cleavage-Based Amplification
- Cycling Probe
Developed by:__
Target Nucleic acid can be either _ OR _
BRANCHED DNA AMPLIFICATION
- Chiron Corp.
- DNA or RNA
the extender probes bind to a __, which in turn bind multiple alkaline phosphatase-labeled nucleotides.
Eight multimers or amplifiers, each with 15 branches, bind to each extender probe bound to the target
first-generation assay
- bDNAamplifier
the extender probes bind preamplifiers, which in turn bind 14–15 amplifiers that can each bind to multiple alkaline phosphatase–labeled .
is added as the substrate for the alkaline phosphatase, and chemiluminescence is measured in a luminometer
- This particular system has a detection limit of about _
second-and third-generation assays
- oligonucleotides
-Dioxetane
- 50 target mol/ml
___ bind to target where probes themselves are amplified.
Probe amplification
- Target-specific synthetic probes
Multiple capture and extender probes can be incorporated that
detect slightly different target sequences as occurs with different isolates of_ and _
BRANCHED DNA AMPLIFICATION
- hepatitis C virus and HIV
- In vivo, this activity is most likely important in repairing DNA
_ has promoted this method as the basis of its Invader system
CLEAVAGE-BASED AMPLIFICATION
- Third Wave Technologies
can serve as template for annealing and ligation of additional primers.
- product of LCR, the LCR is better
classified as probe amplification rather target amplification.
ligated primers
Series of __ are used to capture target nucleic acid, and additional extender probes bind to target nucleic acid, and then to multiple reporter molecule, loading the target nucleic acid with signal.
BRANCHED DNA AMPLIFICATION
- short oligomer probes
The target nucleic acid is released from the cells, the DNA is denatured if DNA is the target, and the target nucleic acid binds to capture probes that are fixed to a solid support
BRANCHED DNA AMPLIFICATION
This forms a nick in the DNA that is extended by _, simultaneously displacing the opposite strand.
The displaced strand is also copied by primers that will
restore the restriction site
The product of this amplification is _
EXPONENTIAL PROBE/TARGET
AMPLIFICATION PHASE
-DNA polymerase
- millions of copies of the initial probe
