MB(ASCP)- Techniques Exam
Questions and Answers 100%
PASS
Probe Amplification Techniques—ANSWER-- LCR (Ligase chain reaction) - generates a signal
by repeated ligation of detection probes complementary to specific sequences in the
template.
- SDA (strand displacement amplification) - isothermal amplification; amplifies probe
product without restriction site due to incorporated modified nucleotides).
- QB replicase (QB) - probes bound to template are amplified by QB replicase.
Transcription-Based Amplification (TBA)—ANSWER--mRNA is the target (RNA signal
amplification)
-TMA and NASBA
-Target RNA-->cDNA--> millions of RNA copies
,-Isothermal process
-Mostly binds to T7 polymerase region (infectious disease)
TMA vs. NASBA—ANSWER-Transcription-mediated Amplification (TMA): 2 enzymes, reverse
transcriptase with RNase activity, RNA polymerase
Nucleic acid sequence-based amplification (NASBA): 3 enzymes, Reverse transcriptase, RNA
polymerase, and RNase H
RNA is target for both. Usually used for infectious disease
Steps of TMA and NASBA—ANSWER-1) Promoter primer binds to RNA target (T7 promoter
region)
2) RT binds and synthesizes a cDNA strand, creating a RNA:DNA hybrid
3) RNase activity of the RT digests the RNA template (RNase H in NASBA)
© 2026 Copyright. All Rights Reserved. This document is
protected by copyright law, Copyrighted By Brittie Donald
,4) Second primer hybridizes to 3' end of the cDNA and RT makes another pass to create
dsDNA
5) T7 RNA polymerase initiates transcription by binding to the T7 promoter sequence of the
dsDNA
Ligase Chain Reaction (LCR)—ANSWER-- Amplifies probes that bind to target sequence
- Detects 1 bp mutations (mutation present= no amplification; mutation absent=
amplification)
- Uses 4 oligonucleotide probes, 2 enzymes (DNA polymerase and DNA ligase), thermocycler
- Steps: (1) Denaturation (2) Annealing: 2 probe sets hybridize together on template and
DNA pol fills in the gap (3) Ligation: gap is closed and ligase will only recognize and ligate
primers that are annealed to the probe
Strand Displacement Amplification (SDA)—ANSWER-- Amplifies probes
-Isothermal
, -4 primers: 2 outer (bumper) primers and 2 inner primers (restriction enzyme recognition
site)
- Stage 1: Target generation ( DNA denatured, outer and inner primers bind close to each
other, DNA polymerase extends using dATPaS, outer probes displace inner primers with
result in 2 probe products)
- Stage 2: Exponential Probe Amplification (Exonuclease added and cuts strand that has
dATPaS, Nick is extended by DNA pol, RE is regenerated and cleaved so it can go to another
recognition site, single-stranded product is amplified and a fluorogenic probe is added to
obtain signal)
Signal Amplification Techniques—ANSWER-- Signal binds to target sequences present in
sample (chemiluminescence)
- Uses Alkaline Phosphatase (ALP) or FRET probe
- Does NOT alter the number of target sequences
- Branched DNA analysis
© 2026 Copyright. All Rights Reserved. This document is
protected by copyright law, Copyrighted By Brittie Donald
Questions and Answers 100%
PASS
Probe Amplification Techniques—ANSWER-- LCR (Ligase chain reaction) - generates a signal
by repeated ligation of detection probes complementary to specific sequences in the
template.
- SDA (strand displacement amplification) - isothermal amplification; amplifies probe
product without restriction site due to incorporated modified nucleotides).
- QB replicase (QB) - probes bound to template are amplified by QB replicase.
Transcription-Based Amplification (TBA)—ANSWER--mRNA is the target (RNA signal
amplification)
-TMA and NASBA
-Target RNA-->cDNA--> millions of RNA copies
,-Isothermal process
-Mostly binds to T7 polymerase region (infectious disease)
TMA vs. NASBA—ANSWER-Transcription-mediated Amplification (TMA): 2 enzymes, reverse
transcriptase with RNase activity, RNA polymerase
Nucleic acid sequence-based amplification (NASBA): 3 enzymes, Reverse transcriptase, RNA
polymerase, and RNase H
RNA is target for both. Usually used for infectious disease
Steps of TMA and NASBA—ANSWER-1) Promoter primer binds to RNA target (T7 promoter
region)
2) RT binds and synthesizes a cDNA strand, creating a RNA:DNA hybrid
3) RNase activity of the RT digests the RNA template (RNase H in NASBA)
© 2026 Copyright. All Rights Reserved. This document is
protected by copyright law, Copyrighted By Brittie Donald
,4) Second primer hybridizes to 3' end of the cDNA and RT makes another pass to create
dsDNA
5) T7 RNA polymerase initiates transcription by binding to the T7 promoter sequence of the
dsDNA
Ligase Chain Reaction (LCR)—ANSWER-- Amplifies probes that bind to target sequence
- Detects 1 bp mutations (mutation present= no amplification; mutation absent=
amplification)
- Uses 4 oligonucleotide probes, 2 enzymes (DNA polymerase and DNA ligase), thermocycler
- Steps: (1) Denaturation (2) Annealing: 2 probe sets hybridize together on template and
DNA pol fills in the gap (3) Ligation: gap is closed and ligase will only recognize and ligate
primers that are annealed to the probe
Strand Displacement Amplification (SDA)—ANSWER-- Amplifies probes
-Isothermal
, -4 primers: 2 outer (bumper) primers and 2 inner primers (restriction enzyme recognition
site)
- Stage 1: Target generation ( DNA denatured, outer and inner primers bind close to each
other, DNA polymerase extends using dATPaS, outer probes displace inner primers with
result in 2 probe products)
- Stage 2: Exponential Probe Amplification (Exonuclease added and cuts strand that has
dATPaS, Nick is extended by DNA pol, RE is regenerated and cleaved so it can go to another
recognition site, single-stranded product is amplified and a fluorogenic probe is added to
obtain signal)
Signal Amplification Techniques—ANSWER-- Signal binds to target sequences present in
sample (chemiluminescence)
- Uses Alkaline Phosphatase (ALP) or FRET probe
- Does NOT alter the number of target sequences
- Branched DNA analysis
© 2026 Copyright. All Rights Reserved. This document is
protected by copyright law, Copyrighted By Brittie Donald
