(0) LMu - - primer primer pair.pcr pair.pcr : Target & Yield DNA Sizes : Target & Yield DNA Sizes

Dual-Dual-Phase PCR Protocol (Phase PCR Protocol (SOPSOP): ):

a)a) Touch-Touch-Down PhaseDown Phase: on original templateson original templates [36.0~30.0oC] x13 cycles, drop 0.5oC each b)b) Steady-Steady-State PhaseState Phase: on PCR yield templateson PCR yield templates

[55.0oC] x23 cycles

Mu-Primer = Total 10 pmol [2/2 or 2/11] run 5/20ul on 1.5% gel M: Size marker of 1 kb plus DNA ladder (Invitrogen Inc.).

((B)) ((B)) PCRPCR

((A)) ((A)) M13R/F

M13R/F Virus

Virus No.No.

592592 0707

0303 TuMV 1757

Poty TuMV 2 Poty

2

982982 1111

0303 ZaMV 1600

Poty ZaMV 5 Poty

5

Potex Potex Carmo Carmo Tombus Tombus Tombus Tombus PotyPoty PotyPoty PotyPoty

Genus Genus

BaMVBaMV HCRSV HCRSV TBSVTBSV CNVCNV ZaMMV ZaMMV PVAPVA DsMVDsMV

Clone Clone

856856 1010

0909 7 4776

7

789789 1111

0303 3 1682

3

99 88 66 44 11 LaneLane

0404 0808 1010 0505 0707

16131613 0101

6366

12081208 0404

3910

856856 0909

4701

916916 0202

1845

414414 0606

1826

SizeSize Primers

Primers SeQSeQ

(0) LMu

(0) LMu - - primer primer pair.pcr pair.pcr : Target & Yield DNA Sizes : Target & Yield DNA Sizes

PCR- PCR -rx on M13 Clones: rx on M13 Clones:

(A) Target

(A) Target Inserts on clones Inserts on clones (B) (B) Dual- Dual -Phase PCR Yield Phase PCR Yield A A

B B

9 8 7 6 M 5 4 3 2 1

(1) LMu

(1) LMu - - primer pair/ primer pair/ ms.pcr ms.pcr : Yield DNA & Specificity : Yield DNA & Specificity

((A,B)) ((A,B)) PCRPCR

M13R/F M13R/F Virus

Virus No.No.

832832 0505

0202 ZaMMV

ZaMMV PotyPoty

33

Potex Potex Carmo Carmo Tombus Tombus Tombus Tombus PotyPoty PotyPoty PotyPoty PotyPoty

Genus Genus

856856 1010

0909 TBSVTBSV

77

592592 0707

0303 TuMVTuMV

22

99 88 66 55 44 11 LaneLane

0404 0808 1010 1111 1111 0707

16131613 0101

BaMVBaMV

12081208 0404

HCRSV HCRSV

856856 0909

CNVCNV

789789 0303

PVAPVA

982982 0303

ZaMVZaMV

414414 0606

DsMVDsMV

SizeSize Primers

Primers Insert

Insert

PCR- PCR -rx on Respective Target: rx on Respective Target:

1) 2 primers pair = 10pmol 1) 2 primers pair = 10pmol

2) 2) All 11 MinSet primers = 10pmol All 11 MinSet primers = 10pmol A A

B B

Dual-Dual-Phase PCR Protocol (Phase PCR Protocol (SOPSOP): ):

a)a) Touch-Touch-Down PhaseDown Phase: on original templateson original templates [36.0~30.0oC] x13 cycles, drop 0.5oC each b)b) Steady-Steady-State PhaseState Phase: on PCR yield templateson PCR yield templates

[55.0oC] x23 cycles

Mu-Primer = Total 10 pmol [2/2 or 2/11] run 5/20ul on 1.5% gel M: Size marker of 1 kb plus DNA ladder (Invitrogen Inc.).

9.BaMV 9.BaMV 7.TBSV

7.TBSV 6.CNV

6.CNV

8.HCRSV 8.HCRSV 4.ZaMV

4.ZaMV 3.ZaMMV

3.ZaMMV

5.PVA 5.PVA 1.DsMV

1.DsMV 2.TuMV

2.TuMV

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●● ●● ●●

●● ●● ●●

(2a) Array

(2a) Array - - hyb hyb *CLE: *CLE: ms.pcr ms.pcr *dig & Ab*ap|CDP *dig & Ab*ap|CDP - - star star

Target:

Target: MS PCR yield with DIGMS PCR yield with DIG--labeledlabeled Probe

Probe--Array: Array: Nine 50Nine 50--mer Arraymer Array--probeprobe PreHyb/Hyb:

PreHyb/Hyb: 42/45oC 1.5/12.0hr with 42/45oC 1.5/12.0hr with Hyb Kit, Roche

Hyb Kit, Roche Wash/Signal:

Wash/Signal: 45/48oC & anti45/48oC & anti--DIG Ab|ap DIG Ab|ap CDP*Kit, NEB

CDP*Kit, NEB Exposure:

Exposure: RToC 16.0hr RToC 16.0hr

(2b) Array

(2b) Array - - hyb hyb *Dye: *Dye: ms.pcr ms.pcr *dig & Ab*ap|NBT- *dig & Ab*ap|NBT - kit kit

9.BaMV 9.BaMV 7.TBSV

7.TBSV 6.CNV

6.CNV

8.HCRSV 8.HCRSV 4.ZaMV

4.ZaMV 3.ZaMMV

3.ZaMMV

5.PVA 5.PVA 1.DsMV

1.DsMV 2.TuMV

2.TuMV

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●● ●● ●●

●● ●● ●●

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●● ●● ●●

●● ●● ●●

●● ●● ●●

Target:

Target: MS PCR yield with DIGMS PCR yield with DIG--labeledlabeled Probe

Probe--Array: Array: Nine 50Nine 50--mer Arraymer Array--probeprobe PreHyb/Hyb:

PreHyb/Hyb: 42/45oC 1.5/12.0hr with 42/45oC 1.5/12.0hr with Hyb Kit, Roche

Hyb Kit, Roche Wash/Signal:

Wash/Signal: 45/48oC & anti45/48oC & anti--DIG Ab|ap DIG Ab|ap NBT*Kit, NEB

NBT*Kit, NEB Exposure:

Exposure: RToC 16.0hr RToC 16.0hr

A

(3) LMu

(3) LMu - - primer pair/ primer pair/ ms.pcr ms.pcr : Target Dose Sensitivity : Target Dose Sensitivity

10pmol All

2 All 2 All 2

All 2

Primer:

fmol 0.1

0.2 1.0

2.0 DNA:

HCRSV PVA

HCRSV PVA

Virus:

M 8 7 6 5 4 3 2 1 M

(A,B) (A,B) PCRPCR

M13R/F M13R/F Virus

Virus No.No.

832832 0505

0202 ZaMMV

ZaMMV PotyPoty

Potex Potex Carmo Carmo Tombus Tombus Tombus Tombus PotyPoty PotyPoty PotyPoty PotyPoty

Genus Genus

856856 1010

0909 TBSVTBSV

592592 0707

0303 TuMVTuMV

3,73,7 1,51,5 LaneLane

0404 0808 1010 1111 1111 0707

16131613 0101

BaMVBaMV

12081208 0404

HCRSV HCRSV

856856 0909

CNVCNV

789789 0303

PVAPVA

982982 0303

ZaMVZaMV

414414 0606

DsMVDsMV

SizeSize Primers

Primers Insert

Insert

Dual-Dual-Phase PCR Protocol (Phase PCR Protocol (SOPSOP): ):

a)a) Touch-Touch-Down PhaseDown Phase: on original templateson original templates [36.0~30.0oC] x13 cycles, drop 0.5oC each b)b) Steady-Steady-State PhaseState Phase: on PCR yield templateson PCR yield templates

[55.0oC] x23 cycles

Mu-Primer = Total 10 pmol [2/2 or 2/11] run 5/20ul on 1.5% gel M: Size marker of 1 kb plus DNA ladder (Invitrogen Inc.).

A B

(3) LMu

(3) LMu - - primer pair/ primer pair/ ms.pcr ms.pcr : Sensitivity & Specificity : Sensitivity & Specificity

10pmol All

2 All 2 All 2

All 2

Primer:

fmol 0.1

0.2 1.0

2.0 DNA:

HCRSV PVA

HCRSV PVA

Virus:

M 8 7 6 5 4 3 2 1

M M 1 2 3 4 5 6 7 8 M

Probe:

Probe: HCRSV 3HCRSV 3’’--DIGDIG--labeled 50labeled 50--mer Arraymer Array--probeprobe PreHyb/Hyb:

PreHyb/Hyb: 42/45oC 1.5/12.0hr with Hyb Kit, Roche 42/45oC 1.5/12.0hr with Hyb Kit, Roche Wash/Signal:

Wash/Signal: 45/48oC & anti45/48oC & anti--DIG Ab|ap CDP*Kit, NEBDIG Ab|ap CDP*Kit, NEB Exposure:

Exposure: RToC 16.0hrRToC 16.0hr