8.4 Isothermal & Target Multiplex Amplification

Q: Which set of enzymes is required to perform Transcription-Mediated Amplification (TMA) in clinical blood donor screening for HIV-1 and HCV?

Reverse Transcriptase (with intrinsic RNase H activity) and T7 RNA Polymerase. Transcription-Mediated Amplification (TMA) is an isothermal nucleic acid amplification technology that operates at a constant 41°C to 42°C using two core enzymes: Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (which possesses both RNA-directed DNA polymerase activity to make cDNA and intrinsic RNase H activity to selectively degrade the initial RNA template strand) and bacteriophage T7 RNA Polymerase. T7 RNA polymerase binds to the double-stranded promoter sequence incorporated by the primers and transcribes 100 to 1,000 single-stranded RNA amplicons per template, achieving over a billion-fold amplification in less than an hour.

Q: A clinical molecular diagnostics laboratory is developing a point-of-care colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay for rapid viral detection. Why does a positive LAMP reaction produce a visual color change from red to yellow when phenol red is used as the indicator in a weakly buffered master mix?

DNA polymerase incorporates hundreds of thousands of dNTPs into growing concatemers, releasing one hydrogen ion (proton, H+) per nucleotide incorporated, which acidifies the reaction and shifts phenol red from red to yellow.. During DNA synthesis, when a DNA polymerase incorporates a deoxynucleotide triphosphate (dNTP) onto the 3' hydroxyl terminus of a growing strand, it releases a molecule of pyrophosphate and a hydrogen ion (proton, H+). Because Loop-Mediated Isothermal Amplification (LAMP) generates enormous quantities of multimeric DNA concatemers (up to 10–50-fold more DNA than conventional PCR), massive quantities of protons are released into solution. In a low-buffering-capacity reaction mix, this influx of protons causes a significant drop in pH (from approximately pH 8.8 down to pH 6.0–6.5), causing the pH indicator dye phenol red to transition from its alkaline pink/red color to an acidic bright yellow.

Q: In large-scale clinical syndromic multiplex PCR panels (such as 20-target respiratory or meningitis panels), what engineering strategy is commonly implemented in nested microfluidic cartridge platforms to eliminate primer-dimer cross-reactivity and prevent reaction component starvation?

Utilizing a two-stage amplification workflow consisting of an initial low-cycle multiplex pre-amplification step followed by microfluidic dilution into individual single-plex nested inner PCR reaction wells. In high-plex syndromic panels containing 20 or more primer pairs, simultaneous amplification in a single bulk tube causes severe primer-primer interactions (hundreds of potential primer-dimer combinations) and reagent starvation, where fast-amplifying targets deplete primers and dNTPs. Advanced closed-cartridge systems (such as the BioFire FilmArray) overcome this by employing a two-stage nested PCR strategy: Stage 1 performs a brief, low-cycle (15–18 cycles) multiplex pre-amplification with outer primers to enrich all targets simultaneously; the reaction is then automatically diluted and distributed across an array of separate microscopic wells for Stage 2, where individual single-plex inner nested primer pairs perform high-cycle real-time PCR and melting analysis without interference from other primers.

Key Takeaways

Isothermal nucleic acid amplification technologies synthesize millions of amplicon copies at a single constant operating temperature (37°C–65°C), eliminating thermal cycling hardware to enable ultra-rapid point-of-care (POC) and high-throughput clinical screening.
Transcription-Mediated Amplification (TMA) and NASBA operate at 41°C–42°C utilizing Reverse Transcriptase, RNase H, and T7 RNA Polymerase to produce 100–1,000 single-stranded RNA amplicons per template cycle, achieving ultra-high sensitivity for blood donor screening (HIV, HCV, HBV) and STIs.
Loop-Mediated Isothermal Amplification (LAMP) operates at 60°C–65°C using 4 to 6 specialized primers and strand-displacing Bst DNA Polymerase to generate multimeric cauliflower-like concatemers detectable via magnesium pyrophosphate turbidity or visual pH/colorimetric shifts.
Strand Displacement Amplification (SDA) utilizes restriction endonuclease hemiphosphorothioate nicking and exo- Bst polymerase, while Helicase-Dependent Amplification (HDA) uses DNA helicase (UvrD) to unwind duplexes enzymatically.
Clinical syndromic multiplex panels (BioFire FilmArray, Luminex xTAG, GenMark ePlex) amplify 15–30+ targets simultaneously, requiring universal-tailed primers, Tm harmonization, and nested microfluidics to prevent primer-dimer interference and reagent depletion.

Last updated: August 2026

8.4 Isothermal & Target Multiplex Amplification

Quick Summary: While conventional and real-time PCR rely on precise thermal cycling between denaturation ($95^\circ\text{C}$), annealing ($55–60^\circ\text{C}$), and extension ($72^\circ\text{C}$), isothermal amplification technologies synthesize massive quantities of nucleic acid amplicons at a single, constant operating temperature ($37^\circ\text{C} – 65^\circ\text{C}$). By replacing thermal denaturation with enzymatic mechanisms—such as T7 RNA transcription, strand-displacing polymerases (Bst), restriction enzyme nicking, or DNA helicases—isothermal systems achieve rapid amplification ($10^9\text{-fold in } 15–45\text{ minutes}$) on simplified, low-power instrumentation ideal for Point-of-Care (POC) diagnostics and ultra-high-throughput clinical screening (Transcription-Mediated Amplification [TMA], LAMP, SDA, HDA, RPA). Simultaneously, multiplex target amplification enables syndromic testing for dozens of respiratory, gastrointestinal, and central nervous system pathogens in a single clinical sample using advanced nested microfluidic cartridge systems.

1. Isothermal Amplification Principles & Point-of-Care Utility

In standard PCR, thermal ramping between temperatures is often the rate-limiting step, requiring complex Peltier blocks, high electrical power, and heated lids. Isothermal amplification overcomes these constraints by operating continuously at a single temperature.

+----------------------------------------------------------------------------------------------------+
|                                ISOTHERMAL vs. THERMOCYCLED AMPLIFICATION                           |
+-------------------+-----------------------------------+--------------------------------------------+
| Parameter         | Thermocycled PCR / qPCR           | Isothermal Amplification (TMA, LAMP, SDA)  |
+-------------------+-----------------------------------+--------------------------------------------+
| **Thermal Profile**| Cyclical: $95^\circ\text{C} \rightarrow 55^\circ\text{C} \rightarrow 72^\circ\text{C}$   | **Constant single temperature** ($41^\circ–65^\circ\text{C}$)   |
+-------------------+-----------------------------------+--------------------------------------------+
| **Strand**        | Thermal heat ($95^\circ\text{C}$) breaks    | **Enzymatic**: RNA degradation (RNase H),  |
| **Separation**    | hydrogen bonds                    | strand displacement (*Bst*), nicking, or   |
|                   |                                   | DNA helicase (*UvrD*)                      |
+-------------------+-----------------------------------+--------------------------------------------+
| **Amplification**  | Binary doubling per cycle         | **Continuous / Geometric transcription**   |
| **Kinetics**      | ($2^n$ accumulation; ~1–2 hours)  | ($10^9$-fold accumulation in 15–40 min)    |
+-------------------+-----------------------------------+--------------------------------------------+
| **Amplicon Nature**| Double-stranded DNA (dsDNA) of    | Single-stranded RNA (TMA/NASBA) or         |
|                   | fixed, discrete base-pair size    | multimeric inverted concatemers (LAMP)     |
+-------------------+-----------------------------------+--------------------------------------------+
| **Instrumentation**| Microprocessor thermal cycler with| Simple heating block, water bath, or       |
|                   | rapid Peltier heating/cooling     | battery-powered POC handheld device        |
+-------------------+-----------------------------------+--------------------------------------------+

2. Transcription-Mediated Amplification (TMA) & NASBA

Transcription-Mediated Amplification (TMA) and Nucleic Acid Sequence-Based Amplification (NASBA) are transcription-based isothermal systems that amplify single-stranded RNA (ssRNA) targets at a constant temperature of $41^\circ\text{C} – 42^\circ\text{C}$.

                         TRANSCRIPTION-MEDIATED AMPLIFICATION (TMA)
                         
   Step 1: Primer 1 (with T7 RNA Polymerase Promoter Tail) binds target ssRNA.
           5'--[ T7 Promoter ]==================>
           ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (Target (+) ssRNA)
                                    |
                                    v (M-MLV Reverse Transcriptase extends cDNA)
           5'--[ T7 Promoter ]========================================== (cDNA strand)
           ------------------------------------------------------------- (Degraded by RNase H!)
                                    |
   Step 2: Primer 2 binds cDNA; RT synthesizes second DNA strand.
                                    <==================== 5' (Primer 2)
           5'--[ T7 Promoter ]==========================================
                                    |
                                    v
           5'==[ ds T7 Promoter ]======================================= (Double-stranded DNA Template)
                                    |
   Step 3: T7 RNA Polymerase binds promoter -> Generates 100 - 1,000 (-) ssRNA transcripts!
                     [ T7 RNA Polymerase ] ===> ===> ===>
           ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
           ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  (100 - 1,000 ssRNA Amplicons
           ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~   per DNA template!)

The Enzymatic Cascade of TMA / NASBA

Enzyme Systems:
- TMA (Hologic Aptima platforms): Uses two enzymes—M-MLV Reverse Transcriptase (possessing both RNA-directed DNA polymerase activity and intrinsic RNase H activity) and T7 RNA Polymerase.
- NASBA (bioMérieux NucliSENS platforms): Uses three enzymes—AMV Reverse Transcriptase, exogenous E. coli RNase H, and T7 RNA Polymerase.
The Amplification Cycle:
1. Primer 1 Binding: A forward primer containing a 5' bacteriophage T7 promoter sequence binds to the target RNA.
2. cDNA Synthesis & Template Degradation: Reverse Transcriptase synthesizes a complementary DNA (cDNA) strand. Intrinsic RNase H selectively degrades the original RNA template from the RNA:DNA heteroduplex.
3. Double-Stranded Promoter Formation: Primer 2 binds the single-stranded cDNA, and Reverse Transcriptase extends it to create a fully double-stranded DNA template containing an active, functional double-stranded T7 promoter.
4. High-Yield Autocatalytic Transcription: T7 RNA polymerase binds the promoter and transcribes $100\text{ to } 1,000\text{ single-stranded RNA amplicons}$ from each DNA template. Each newly synthesized RNA amplicon immediately enters the cycle as a template for Primer 2, driving exponential amplification that yields $>10^9$-fold amplification in under 45 minutes.

Clinical Diagnostic Applications of TMA

Blood Donor Screening: Ultra-sensitive screening of pooled donor blood for HIV-1 RNA, HCV RNA, HBV DNA, West Nile Virus (WNV), and Zika Virus.
Sexually Transmitted Infections (STIs): Gold standard testing on the Hologic Panther system for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium.
Oncology: Detection of oncogenic HPV E6/E7 mRNA transcripts, which correlate with active cervical dysplasia more specifically than HPV DNA testing.

3. Loop-Mediated Isothermal Amplification (LAMP)

Loop-Mediated Isothermal Amplification (LAMP) is an ultra-high-efficiency isothermal technology operating at $60^\circ\text{C} – 65^\circ\text{C}$ that produces up to $10^{10}$ amplicon copies within $20–30\text{ minutes}$.

                             LAMP PRIMER DESIGN & TARGET REGIONS
                             
                 [ F3c ]    [ F2c ]    [ F1c ]          [ B1 ]    [ B2 ]    [ B3 ]
       5' --------+----------+----------+----------------+---------+---------+-------- 3'
       3' --------+----------+----------+----------------+---------+---------+-------- 5'
                 [ F3  ]    [ F2  ]    [ F1  ]          [ B1c]    [ B2c]    [ B3c]
       
       Primers:
         • FIP (Forward Inner Primer):  [ F1c sequence ] + [ F2 sequence ]
         • BIP (Backward Inner Primer): [ B1c sequence ] + [ B2 sequence ]
         • F3 (Forward Outer Primer):   Displaces FIP-extended strand
         • B3 (Backward Outer Primer):  Displaces BIP-extended strand
         • Loop Primers (LoopF / LoopB): Accelerate amplification by priming stem loops

The Mechanism of LAMP

Enzyme: Utilizes Bst DNA Polymerase (Large Fragment) purified from Bacillus stearothermophilus, which possesses powerful $5' \rightarrow 3'$ strand-displacement activity and lacks $5' \rightarrow 3'$ exonuclease activity.
Primer Design Architecture: Employs 4 to 6 specialized primers recognizing 6 to 8 distinct regions on the target sequence:
- Forward Inner Primer (FIP): Hybridizes via its F2 region. Its 5' F1c tail is complementary to the downstream F1 region.
- Outer Primers (F3 and B3): Anneal upstream of the inner primers; extension by Bst displaces the newly synthesized FIP/BIP strands.
- Dumbbell Stem-Loop Formation: The displaced single-stranded DNA folds back on itself as F1c pairs with F1 (and B1c with B1), creating a self-annealing dumbbell-shaped structure.
- Concatemer Synthesis: The 3' end of the dumbbell serves as a self-primer for continuous elongation, generating large, repeating multimeric cauliflower-like concatemers of alternating inverted repeats.
- Loop Primers (LoopF and LoopB): Bind to loops not involved in self-priming, speeding up the reaction from 60 minutes down to $15–20\text{ minutes}$.

                             DUMBBELL STRUCTURE IN LAMP
                             
                              /---\               /---\
                             /     \             /     \
                            |  F1c  |===========|  B1   |
                             \     /             \     /
                              \---/ (F1)     (B1c)\---/

Detection Chemistries for LAMP

Magnesium Pyrophosphate Turbidity: As Bst incorporates hundreds of thousands of dNTPs, massive quantities of inorganic pyrophosphate ($\text{P}_2\text{O}_7^{4-}$) are released, reacting with $\text{Mg}^{2+}$ in the buffer to precipitate insoluble magnesium pyrophosphate ($\text{Mg}_2\text{P}_2\text{O}_7$), producing visible white turbidity measured in real time.
Metal-Chelating Dyes (Calcein / Hydroxynaphthol Blue [HNB]): Calcein fluorescence is initially quenched by binding manganese ($\text{Mn}^{2+}$). As pyrophosphate is generated, it strips $\text{Mn}^{2+}$ from calcein, allowing it to bind $\text{Mg}^{2+}$ and emit bright visual green fluorescence.
pH-Sensitive Colorimetric Shifts (Phenol Red): DNA polymerase extension releases one proton ($\text{H}^+$) per incorporated nucleotide ($\text{dNTP} \rightarrow \text{dNMP} + \text{PPi} + \text{H}^+$). In a weakly buffered master mix, the release of millions of protons drops the reaction pH from $8.8$ to $<6.5$, inducing an instantaneous visual color change in phenol red from pink/red (negative) to bright yellow (positive).

4. Other Isothermal Technologies: SDA, HDA & RPA

+----------------------------------------------------------------------------------------------------+
|                                 SDA, HDA & RPA COMPARISON                                          |
+-------------------+--------------------+--------------------+--------------------------------------+
| Isothermal Assay  | Operating Temp     | Primary Enzymes    | Molecular Mechanism & Clinical Assay |
+-------------------+--------------------+--------------------+--------------------------------------+
| **Strand**        | $37^\circ–52^\circ\text{C}$| • Restriction      | Primers incorporate a hemiphosphoro- |
| **Displacement**  | (Thermophilic SDA  |   Endonuclease     | thioate restriction site (*HincII*). |
| **Amplification** | at $52^\circ–55^\circ\text{C}$) |   (*HincII* or *BsoBI*)| Enzyme nicks un-modified strand;      |
| **(SDA)**         |                    | • *exo- Bst* DNA   | *exo- Bst* extends from nick,        |
|                   |                    |   Polymerase       | displacing downstream strand.        |
|                   |                    | • $\text{dATP}\alpha\text{S}$ modified| **Clinical: BD Viper (CT/NG)**       |
+-------------------+--------------------+--------------------+--------------------------------------+
| **Helicase-**     | $60^\circ–65^\circ\text{C}$| • Thermostable DNA | A DNA helicase (*UvrD*) unwinds the  |
| **Dependent**     | (Thermophilic tHDA)|   Helicase (*UvrD*) | DNA duplex enzymatically, eliminating|
| **Amplification** |                    | • Strand-displacing| thermal denaturation. Primers bind   |
| **(HDA)**         |                    |   DNA Polymerase   | ssDNA; polymerase extends.           |
|                   |                    | • Accessory cofactors| **Clinical: Quidel Solana / AmpliVue**|
+-------------------+--------------------+--------------------+--------------------------------------+
| **Recombinase**   | **$37^\circ–42^\circ\text{C}$**| • T4 Recombinase   | Recombinase (UvsX) + UvsY loads onto |
| **Polymerase**    | (Body temperature; |   (UvsX / UvsY)    | primers, scanning dsDNA to invade    |
| **Amplification** | rapid 15-min POC)  | • Single-Stranded  | homologous duplexes. *Bsu* polymerase|
| **(RPA)**         |                    |   Binding (gp32)   | extends; SSB stabilizes D-loop.      |
|                   |                    | • *Bsu* DNA Pol    | **Clinical: Lateral flow POC strips**|
+-------------------+--------------------+--------------------+--------------------------------------+

5. Target Multiplex Amplification & Syndromic Panel Design

Clinical diagnostic microbiology has transitioned toward syndromic multiplex testing, simultaneously interrogating 15 to 30+ potential pathogens from a single clinical specimen (e.g., Upper Respiratory Panels, Gastrointestinal Panels, Meningitis/Encephalitis Panels).

                             SYNDROMIC MULTIPLEX PANEL ARCHITECTURE
                             
      Clinical Specimen (e.g., Nasopharyngeal Swab in VTM)
           |
           v
      [ High-Plex Microfluidic Cartridge (e.g., BioFire FilmArray) ]
           +----------------------------------------------------------------------+
           | Stage 1: Low-Cycle Multiplex Pre-Amplification (Outer Primers)      |
           |          Enriches all 20+ viral/bacterial targets simultaneously     |
           +----------------------------------+-----------------------------------+
                                              |
                                              v  (Aliquoted into micro-wells)
           +----------------------------------+-----------------------------------+
           | Stage 2: Single-Plex High-Resolution Inner Nested PCR                |
           |          Micro-well 1: Influenza A      Micro-well 5: SARS-CoV-2     |
           |          Micro-well 2: Influenza B      Micro-well 6: RSV A/B        |
           |          Micro-well 3: Adenovirus       Micro-well 7: Human Metapneum|
           |          Micro-well 4: Rhinovirus       Micro-well 8: Bordetella pert|
           +----------------------------------------------------------------------+

The Mathematics & Challenges of Multiplex Primer Design

Primer Interaction Formula: In a multiplex assay containing $N$ individual primer pairs ($2N$ total primer oligonucleotides), the number of potential pairwise primer-primer interactions ($I$) scales quadratically: $I = \frac{2N(2N - 1)}{2} = N(2N - 1)$ For a 20-target panel (40 primers), there are $40(39)/2 = 780\text{ potential primer-primer cross-hybridizations}$ that can form non-specific primer-dimers!
Amplicon Competition & Bias: Amplicons with lower GC content, shorter lengths, or higher initial template concentrations amplify faster, rapidly exhausting master mix dNTPs and $\text{Mg}^{2+}$ cations while starving longer or GC-rich targets.

Multiplex Optimization Strategies

$T_m$ Harmonization: All primer melting temperatures must be strictly matched within $\pm 1.0^\circ\text{C}$ (typically $58^\circ–60^\circ\text{C}$).
Universal-Tailed Primers: Primers are synthesized with a target-specific 3' sequence and a common universal 5' tail sequence. After the first two cycles, all amplicons incorporate the universal tail, allowing subsequent cycles to be driven by a single universal primer pair, equalizing amplification efficiencies across all targets.
Two-Stage Nested Microfluidics (BioFire FilmArray):
- Stage 1 (Multiplex Outer PCR): A low-cycle (15–18 cycles) bulk multiplex PCR enriches all targets using outer primer pairs.
- Stage 2 (Nested Inner Single-Plex PCR): The diluted Stage 1 product is pumped across an array of hundreds of individual micro-wells, each containing a single inner nested primer pair for real-time fluorescence and melt curve identification.

6. Comprehensive Comparative Synthesis of Isothermal Platforms

Platform / Assay	Operating Temp	Enzymes Employed	Primers Required	Amplicon Type	Primary Clinical Diagnostic Tests
Transcription-Mediated Amplification (TMA)	$41^\circ–42^\circ\text{C}$	M-MLV RT (RNase H) + T7 RNA Polymerase	2 (Primer 1 with T7 promoter tail)	Single-stranded RNA ($100–1,000\text{ copies/cycle}$)	*Blood screening (HIV/HCV/HBV/WNV), C. trachomatis, N. gonorrhoeae, HPV mRNA*
Nucleic Acid Sequence-Based Amplification (NASBA)	$41^\circ\text{C}$	AMV RT + E. coli RNase H + T7 RNA Polymerase	2 (T7 promoter primer)	Single-stranded RNA	Enterovirus, CMV pp67 mRNA, respiratory viral loads
Loop-Mediated Isothermal Amplification (LAMP)	$60^\circ–65^\circ\text{C}$	Strand-displacing Bst DNA Polymerase	4 to 6 (FIP, BIP, F3, B3, LoopF, LoopB)	Multimeric concatemers with stem loops	Rapid POC SARS-CoV-2, malaria, tuberculosis, norovirus
Strand Displacement Amplification (SDA)	$37^\circ–52^\circ\text{C}$	Restriction Endonuclease (HincII) + exo- Bst Pol	4 (2 bumper + 2 nicking primers)	Single-stranded DNA fragments	BD Viper C. trachomatis / N. gonorrhoeae
Helicase-Dependent Amplification (HDA)	$60^\circ–65^\circ\text{C}$	DNA Helicase (UvrD) + Strand-displacing DNA Pol	2 standard primers	Double-stranded DNA amplicons	*Quidel Solana / AmpliVue C. difficile, Group A Strep, HSV 1/2*
Recombinase Polymerase Amplification (RPA)	$37^\circ–42^\circ\text{C}$	T4 UvsX / UvsY + gp32 SSB + Bsu DNA Polymerase	2 standard primers	Double-stranded DNA amplicons	Handheld field diagnostics, Ebola, Dengue, biothreat pathogen detection

Loading diagram...

Isothermal Amplification Mechanisms: TMA vs LAMP vs SDA

Test Your Knowledge

Which set of enzymes is required to perform Transcription-Mediated Amplification (TMA) in clinical blood donor screening for HIV-1 and HCV?

Taq DNA Polymerase, Uracil-DNA Glycosylase (UDG), and DNA Helicase

Reverse Transcriptase (with intrinsic RNase H activity) and T7 RNA Polymerase

Bst DNA Polymerase (Large Fragment), DNA Ligase, and Exonuclease III

Restriction endonuclease HincII, terminal transferase, and alkaline phosphatase

Test Your Knowledge

A clinical molecular diagnostics laboratory is developing a point-of-care colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay for rapid viral detection. Why does a positive LAMP reaction produce a visual color change from red to yellow when phenol red is used as the indicator in a weakly buffered master mix?

Bst DNA polymerase consumes all free calcium cations in the buffer, elevating the pH above 10.0.

The reaction generates pyrophosphate crystals that absorb green light and reflect yellow wavelengths.

DNA polymerase incorporates hundreds of thousands of dNTPs into growing concatemers, releasing one hydrogen ion (proton, H+) per nucleotide incorporated, which acidifies the reaction and shifts phenol red from red to yellow.

Phenol red intercalates into the minor groove of dumbbell-shaped DNA stem loops, altering its absorbance spectrum.

Test Your Knowledge

In large-scale clinical syndromic multiplex PCR panels (such as 20-target respiratory or meningitis panels), what engineering strategy is commonly implemented in nested microfluidic cartridge platforms to eliminate primer-dimer cross-reactivity and prevent reaction component starvation?

Performing all 40 primer extensions at 95°C without any cooling steps

Adding 50% formamide to completely prevent non-specific hydrogen bonding between primers

Replacing all dNTPs with ribonucleotide triphosphates (rNTPs) to speed up Taq polymerase

Utilizing a two-stage amplification workflow consisting of an initial low-cycle multiplex pre-amplification step followed by microfluidic dilution into individual single-plex nested inner PCR reaction wells

Up Next

9.1 Sanger Dideoxy Sequencing & Electropherogram Interpretation

DNA Sequencing Technologies

ASCP Molecular Biology Technologist

ASCP Molecular Biology Technologist

8.4 Isothermal & Target Multiplex Amplification

Key Takeaways

8.4 Isothermal & Target Multiplex Amplification

1. Isothermal Amplification Principles & Point-of-Care Utility

2. Transcription-Mediated Amplification (TMA) & NASBA

The Enzymatic Cascade of TMA / NASBA

Clinical Diagnostic Applications of TMA

3. Loop-Mediated Isothermal Amplification (LAMP)

The Mechanism of LAMP

Detection Chemistries for LAMP

4. Other Isothermal Technologies: SDA, HDA & RPA

5. Target Multiplex Amplification & Syndromic Panel Design

The Mathematics & Challenges of Multiplex Primer Design

Multiplex Optimization Strategies

6. Comprehensive Comparative Synthesis of Isothermal Platforms

ASCP Molecular Biology Technologist

1Introduction & Exam Strategy

2Nucleic Acid Chemistry & Molecular Structure

3Central Dogma, Genome Organization & Cytogenetics

4Biochemical Reagents & Enzymes

5Nucleic Acid Isolation & Purification

6Nucleic Acid Quantification & Electrophoresis

7PCR Principles & Primer Design Optimization

8Advanced Amplification Technologies

9DNA Sequencing Technologies

10Hybridization, Blotting & Cytogenetic Assays

11Molecular Infectious Disease Diagnostics

12Molecular Oncology & Hematopathology

13Medical Genetics, Pharmacogenomics & Identity Testing

14Quality Assurance, Controls & Method Validation

15Laboratory Operations, Safety & Clinical Variant Reporting

ASCP Molecular Biology Technologist

8.4 Isothermal & Target Multiplex Amplification

Key Takeaways

8.4 Isothermal & Target Multiplex Amplification

1. Isothermal Amplification Principles & Point-of-Care Utility

2. Transcription-Mediated Amplification (TMA) & NASBA

The Enzymatic Cascade of TMA / NASBA

Clinical Diagnostic Applications of TMA

3. Loop-Mediated Isothermal Amplification (LAMP)

The Mechanism of LAMP

Detection Chemistries for LAMP

4. Other Isothermal Technologies: SDA, HDA & RPA

5. Target Multiplex Amplification & Syndromic Panel Design

The Mathematics & Challenges of Multiplex Primer Design

Multiplex Optimization Strategies

6. Comprehensive Comparative Synthesis of Isothermal Platforms