NUCLEIC ACID AMPLIFICATION TECHNIQUES II

Question 1

• Number of target nucleic acid sequences in the sample is not changed compared to the target amplification procedure, such as __.

Answer 1

PROBE AMPLIFICATION
- PCR

Question 2

THREE MAJOR PROCEDURES of PROBE AMPLIFICATION

Answer 2

• Ligase Chain Reaction
• Standard Displacement Amplification
• QB Replicase

Question 3

▪ Manufactured and marketed by Abbott
- commercially available

Answer 3

Ligase Chain Reaction

Question 4

▪ Manufactured and marketed by Beckton Dickinson
- commercially available

Answer 4

Standard Displacement Amplification

Question 5

▪ Manufactured and marketed by Vysis
- commercially available.

Answer 5

QB Replicase

Question 6

• 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 __.

Answer 6

• LIGASE CHAIN REACTION (LCR)
• oligonucleotide primers

Question 7

Less risk of carryover contamination resulting in a positive test in bDNA assay than in the __.

Answer 7

BRANCHED DNA AMPLIFICATION
PCR

Question 8

• is currently available for the
  qualitative and quantitative detection of _, _, _

Answer 8

• bDNA signal amplification assay
• Hep B, Hep C, and HIV 1

Question 9

Developed by _
Used primarily for the detection of molecular characterization of _ in Genitourinary specimens
Also available for the detection of _ and _

Answer 9

HYBRID CAPTURE ASSAYS
- DigeneDiagnostics
- Human Papilloma Virus (HPV)
- Hepatitis B and Cytomegalovirus (CMV)

Question 10

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

Answer 10

HYBRID CAPTURE ASSAYS
- 1000
- DNA/RNA Hybrid

Question 11

Captured hybrids are detected by binding of__ in a typical sandwich assay
The substrate for the alkaline
phosphatase is added, and
_ is measured

Answer 11

• alkaline phosphate conjugated anti-DNA/RNA hybrid antibodies
• chemiluminescence

Question 12

Detects target nucleic acids by using a series of probes that bind to the target and overlap
- Target is __ primarily __

Answer 12

CLEAVAGE-BASED AMPLIFICATION
- DNA polymorphisms ( Factor V Leiden mutations)

Question 13

–Enzyme isolated from bacteria that recognizes overlapping
sequences of DNA and makes a cut in the overlapping piece

Answer 13

Cleavase

Question 14

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

Answer 14

CYCLING PROBE
- Methicillin resistance (mecA) in Staphylococcus aureus
o Vancomycin resistance (vanAand vanB) in
Enterococcus

Question 15

METHODS OF PROBE AMPLIFICATION

Answer 15

1. Branched DNA Amplification
2. Hybrid Capture Assay
3. Cleavage-Based Amplification
4. Cycling Probe

Question 16

• In LCR, ___________is used to ligate the adjacent primers together.

Answer 16

dna ligase

Question 17

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.

Answer 17

LCR
- beta globulin
- sickle cell disease

Question 18

It differs from most of the previous methods, it is an isothermal amplification process.
It is more similar to LCR than PCR

Answer 18

STRAND DISPLACEMENT
AMPLIFICATION (SDA)

Question 19

There are two stages to SDA process:

Answer 19

Target Generation Phase
Exponential Probe or Target Amplification Phase

Question 20

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 __

Answer 20

. TARGET GENERATION PHASE
- 95°C
- restriction enzyme

Question 21

_ extends the primers, incorporating a modified nucleotide,_

Answer 21

Exonuclease- deficient DNA polymerase derived from E. coli
- 2′ deoxyadenosine 5′-O-(1-thiotriphosphate) (dATPaS)

Question 22

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.

Answer 22

EXPONENTIAL PROBE/TARGET
AMPLIFICATION PHASE
- dATPaS

Question 23

APPLICATION
Detection of , AND _
Addition of a _ to the reaction produces a fluorescent signal that corresponds to the amount of amplified target.

Answer 23

EXPONENTIAL PROBE/TARGET
AMPLIFICATION PHASE

M. tuberculosis, C. trachomatis, N. gonorrhoeae
- fluorogenic probe

Question 24

Method for amplifying probes that have specificity for a target sequence.
Named for the major enzyme that is used to amplify probe sequences.

Answer 24

Qβ REPLICASE

Question 25

RNA-dependent RNA polymerase from the bacteriophage Qβ.

Answer 25

Qβ REPLICASE

Question 26

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.

Answer 26

Qβ replicase - reporter probes

Question 27

–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 _

Answer 27

Reporter probes - (midivariant-1) - 10^6–10^9 RNA molecules/probe - 15 minutes

Question 28

APPLICATION of Reporter probes Used primarily to amplify nucleic associated w/ infectious organisms:

Answer 28

Mycobacteria Chlamydia HIV CMV

Question 29

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

Answer 29

SIGNAL AMPLIFICATION

Question 30

SIGNAL AMPLIFICATION METHODS: 1 2 3 4

Answer 30

1. * Branched DNA Amplification 2. * Hybrid Capture Assay 3. * Cleavage-Based Amplification 4. * Cycling Probe

Question 31

Developed by:__ Target Nucleic acid can be either _ OR _

Answer 31

BRANCHED DNA AMPLIFICATION - Chiron Corp. - DNA or RNA

Question 32

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

Answer 32

first-generation assay - bDNAamplifier

Question 33

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 _

Answer 33

second-and third-generation assays - oligonucleotides -Dioxetane - 50 target mol/ml

Question 34

___ bind to target where probes themselves are amplified.

Answer 34

Probe amplification - Target-specific synthetic probes

Question 35

Multiple capture and extender probes can be incorporated that detect slightly different target sequences as occurs with different isolates of_ and _

Answer 35

BRANCHED DNA AMPLIFICATION - hepatitis C virus and HIV

Question 36

- In vivo, this activity is most likely important in repairing DNA _ has promoted this method as the basis of its Invader system

Answer 36

CLEAVAGE-BASED AMPLIFICATION - Third Wave Technologies

Question 37

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.

Answer 37

ligated primers

Question 38

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.

Answer 38

BRANCHED DNA AMPLIFICATION - short oligomer probes

Question 39

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

Answer 39

BRANCHED DNA AMPLIFICATION

Question 40

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 _

Answer 40

EXPONENTIAL PROBE/TARGET AMPLIFICATION PHASE -DNA polymerase - millions of copies of the initial probe