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Summary of PCR performance of blended DNA polymerases (systems) tested in this study_
| TaKaRa Ex Taq™ combined with DreamTaq™ in TaKaRa Ex buffer | PicoMaxx™ System combined with DreamTaq™ in PicoMaxx™ buffer | DreamTaq™ in PicoMaxx™ buffer | pfu in PicoMaxx™ buffer | Pfu combined with DreamTaq™ in PicoMaxx™ buffer | pfu combined with DreamTaq™ in DreamTaq™ buffer | pfu combined with DreamTaq™ in pfu buffer | Pwo in PicoMaxx™ buffer | Pwo combined with DreamTaq™ in Pwo buffer | Pwo combined with DreamTaq™ in PicoMaxx buffer | |
|---|---|---|---|---|---|---|---|---|---|---|
| Spring specimens | na | na | na | na | na | na | na | na | na | na |
| Summer specimens | 3.5 kb: X | 3.5 kb: √√√/X | 3.5 kb: √/X or X | 3.5 kb: X | 3.5 kb: √√√√ or √√ | 3.5 kb: X | 3.5 kb: X | 3.5 kb: X | 3.5 kb: X (DNS) | 3.5 kb: √/X |
| Fall specimens | na | na | na | na | na | na | na | na | na | na |
Summary of PCR performance of Individual DNA polymerases (systems) tested in this study_
| Platinum™ Taq | Taq2000™ | DreamTaq™ | TaKaRa Ex Taq™ | PicoMaxx™ System | pfu | Pwo | Herculase® II | Phusion™ | |
|---|---|---|---|---|---|---|---|---|---|
| Spring specimens | 3.5 kb: X; 1.9 kb: √/X | 3.5 kb: X; 1.9 kb: √√√√/X | 3.5 kb: X; 1.9 kb: √√√√ | na | na | na | na | na | na |
| Summer specimens | na | 3.5 kb: X (DNS); 1.9 kb: √√√ (DNS) | 3.5 kb: X or X/√ (DNS); 1.7Kb: √√√√/X; 1.9 kb: √√√√/X | 3.5 kb: √√/X or X | 3.5 kb: √√/X or X | 3.5 kb: X (DNS) | 3.5 kb: X (DNS) | 3.5 kb: X | 3.5 kb: X |
| Fall specimens | na | na | 3.5 kb: √√√/X; 1.9 kb: NA | na | na | na | na | na | na |
Litylenchus crenatae specimens from American beech trees (Fagus grandifolia) with BLD tested in this study_
| Specimens | Locality | Part | Session |
|---|---|---|---|
| 104H78, 104H81, 104H82, 104H83, 104H84, 104H85, 104H86, 104H87, 104H88, 104H89 and 104H90 | Lake County, Ohio | Leaf | Fall (November, 2017) |
| 104J54, 104J55, 104J56 and 104J57 | Cuyahoga County, Ohio | Leaf | Summer (May, 2018) |
| 104K17, 104K18, 104K19 and 104K20 | The Holden Arboretum, Kirtland, Ohio | Leaf | Summer (August, 2018) |
| 104K25, 104K26, 104K27, 104K28, 104K29, 104K30 and 104K31 | Potter County, Pennsylvania | Leaf | Summer (August, 2018) |
| 104K37, 104K38 and 104K39 | Crawford County, Pennsylvania | Leaf | Summer (August, 2018) |
| 104N95, 104N96 and 104N97 | The Holden Arboretum, Kirtland, Ohio | Bud | Spring (March, 2019) |
PCR cycling conditions_
| No. Step | pfu or combined with DreamTaq™ | Herculase® II | Phusion™ | Pwo or combined with DreamTaq™ | ||||
|---|---|---|---|---|---|---|---|---|
| 1. Initial denaturation | 95°C for 2 min | Step 1: 1 cycle | 95°C for 2 min | Step 1: 1 cycle | 95°C for 2 min | Step 1: 1 cycle | 95°C for 2 min | Step 1: 1 cycle |
| 2. Denaturation | 95°C for 30 sec | Step 2, 3 and 4: 36 cycles | 95°C for 20 sec | Step 2, 3 and 4: 36 cycles | 95°C for 20 sec | Step 2, 3 and 4: 36 cycles | 95°C for 30 sec | Step 2, 3 and 4: 36 cycles |
| 3. Annealling | 55°C for 45 sec | 55°C for 20 sec | 55°C for 20 sec | 57°C for 45 sec | ||||
| 4. Extension | 72°C for 5 min | 72°C for 2 min 15 sec | 72°C for 2 min 15 sec | 72°C for 5 min | ||||
| 5. Final extension | 72°C for 7 min | Step 5: 1 cycle | 72°C for 7 min | Step 5: 1 cycle | 72°C for 7 min | Step 5: 1 cycle | 72°C for 7 min | Step 5: 1 cycle |
PCR components and setup_
| Platinum™ Taq (10 units/μl) | Taq2000™ (5 units/μl) | DreamTaq™ (5 units/μl) | TaKaRa Ex Taq™ (5 units/μl) or combined with DreamTaq™ (5 units/μl) | PicoMaxx™ System (5 units/μl) or combined with DreamTaq™ (5 units/μl) | pfu DNA polymerase (2.5 units/μl) or combined with DreamTaq™ (5 units/μl) | Herculase® II Fusion DNA polymerase | Phusion™ High-Fidelity DNA Polymerase (2 units/μl) | Pwo DNA polymerase (5 units/μl) or combined with DreamTaq™ (5 units/μl) | |
|---|---|---|---|---|---|---|---|---|---|
| Water (μl) | Mixture A: 7.6 | ||||||||
| Water (μl) | 15.375 | 16 | 16.375/16.25 | 15.875 or 15.75 | 17.3 or 17.175 | 17.05/17.55 or 16.925/17.425 | 16 | 14.5 | Mixture B: 9.875 (or 9.75) |
| 10 or 5x proprietary buffer (μl) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 5 | 5 | Mixture A: 2.5 |
| 50 mM MgCl2 (μl) | 1 | 0.25 | |||||||
| 100 mM dNTP (25 mM each) (μl) | 0.2 | 0.2 | 0.25 | Mixture B: 0.4 | |||||
| 10 mM dNTP (2.5 mM each) (μl) | 2 | 0.5 | |||||||
| 8 mM dNTP (2 mM each) (μl) | 2.5 | 2.5 | 2.5 | ||||||
| 10 μm Forward primer (μl) | 0.75 | 0.75 | 0.75 | 1.25 | 1.25 | 1.25 | 0.625 | 1.25 | Mixture B: 1.25 |
| 10 μm Reverse primer (μl) | 0.75 | 0.75 | 0.75 | 1.25 | 1.25 | 1.25 | 0.625 | 1.25 | Mixture B: 1.25 |
| DMSO | 0.25 | ||||||||
| DNA template (μl) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | Mixture B: 2 |
| Proprietary DNA polymerase(s) (μl) | 0.125 | 0.25 | 0.125/0.25 | 0.125 or plus DreamTaq™: 0.125 | 0.5 or plus DreamTaq™: 0.125 | 0.75/0.25 or plus DreamTaq™: 0.125 | 0.5 | 0.25 | Mixture A: 0.125 or plus DreamTaq™: 0.125 |
| Total reaction volume (μl) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 12.5 each mixture |