1KbcutB orf5-3orf2cutC cutA orf3 orf4orf1 orf6

cutB orf5-1orf2cutC cutA orf3 orf4cutR

5 : Primer 5F and 6R

Primer 1F : orf1-cutB RT-FPrimer BR : orf1-cutB RT-RPrimer 3F : orf3del-FPrimer 4R : orf4del-RPrimer 4F : orf4del-FPrimer 5R : orf5del-RPrimer 5F : copy2-P3Primer 6R : copy2-P2

1 : Primer 1F and BR : 61 ℃ 2 : Primer 3F and 5R : 61 ℃ 3 : Primer 3F and 4R : 61 ℃ 4 : Primer 4F and 5R : 61 ℃ 5 : Primer 5F and 6R : 61℃

4 : Primer 4F and 5R

3: Primer 3F and 4R 413 bp

2 : Primer 3F and 5R

1 : Primer 1F and BR

SM 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

RT-PCR cDNA(O) RT-PCR cDNA(X) PCR

366 bp13641433

15601512

RT-PCR protocol1. Prepare 5 ㎍ of RNA and add DNase I (50U) in 8 ㎕ of RNase-free water a

nd add 2 ㎕ of 5X Superscript II buffer (invitrogen) and incubate at 37 for ℃1h.

2. Add 1 ㎕ of primer (0.5 pmol/ ㎕ ) and heat 70 for 10 min.℃3. Cool on ice.4. Add 4 ㎕ of 10 mM dNTP, 1 ㎕ of 0.1M DTT, 2 ㎕ of 5X Superscript II

buffer, 2 ㎕ of RNase-free water and 1 ㎕ of reverse transcriptase (200U).5. Incubate at 42 for 1 h.℃6. Heat the tube 90 for 10 min.℃7. Go to RT-PCR. A 2 3 4 A 2 3 4