Quy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vậtQuy trình thiết kế pcr môn chẩn đoán bệnh động vật
Trang 1Biến tính:94-95oC/2-4p; Bắt cặp:66-67oC/30s; Kéo dài:72oC/45s; Kéo dài cuối:72oC/5-10p
3 Thiết kế cặp primer và giới thiệu enzyme cắt vào gen mục tiêu
NHeI-ORF2 FOR: 5’atatatgctagcATGACGTATCCAAGGAGGCG3’(20nt, 67oC)
KpnI-ORF2 REV: 5’atatatgttaacTTAAGGGTTAAGTGGGGGGT3’
(Orginal seq:5’ACCCCCCACTTAACCCTTAAgttaacatatat3’)(66oC)
3.1 Tạo cặp primer giới thiệu HA tag vào
ORF2:TACCCATACGATGTTCCAGATTACGCTđặt vào đầu 3’của gene
NHeI-ORF2 FOR: 5’atatatgctagcATGACGTATCCAAGGAGGCG3’(20nt, 67oC)
KpnI-ORF2+HA REV: 5’atatatgttaacTTAAGGGTTAAGTGGGGGG3’=> Dùng so sánh
5’atatgttaacTTAAGCGTAATCTGGAACATCGTATGGGTAAGGGTTAAGTGGGGGGT3’ (17nt,66oC)(Orginal seq:5’ACCCCCCACTTAACCCTTACCCATACGATGTTCCAGATTACGCTTAAgttaacatatat3’)
3.2 Tạo cặp primer giới thiệu HA tag vào
ORF2:TACCCATACGATGTTCCAGATTACGCTđặt vào đầu 5’của gene
NHeI-ORF2+HA FOR:
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTACGTATCCAAGGAGGCGTTAC3’(20nt,65oC)
KpnI-ORF2 REV: 5’atatgttaacTTAAGGGTTAAGTGGGGGGT3’(66oC)
(Orginal seq:5’ACCCCCCACTTAACCCTTAAgttaacatatat3’)
35 chu kỳ:
Biến tính:94-95oC/2-4p; Bắt cặp:66-67oC/30s; Kéo dài:72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: ORF2 gene or PCV2 genome
Phần 1: Thiết kế cặp primer hoặc tham khảo primer có sẵn
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACATGCCCAGCA 60OR537692.1
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACATGCCCAGCA 60 ************************************************************
Trang 2240 ************************************* **********************
Trang 3900OR537692.1 CCTCAACTGCTGTCCCAGCTGTAGAAGCTCTTTATCGGAGGATTACTTCCTTGGTATTTT
900 ************************************************************
KT719404.1
GGAAGAATGCTACAGAACAATCCACGGAGGAAGGGGGCCAGTTCGTCACCCTTTCCCCCC 960
Trang 41020OR537692.1 CATGCCCTGAATTTCCATATGAAATAAATTACTGAGTCTTTTTTATCACTTCGTAATGGT
1020 ************************************************************
KT719404.1 TTTTATTATTCATTAAGGGTTAAGTGGGGGGTCTTTAAGGTTAAATTCTCTGAATTGTAC
1080OR537693.1 TTTTATTATTCATTAAGGGTTAAGTGGGGGGTCTTTAAGATTAAATTCTCTGAATTGTAC
1080OR537692.1 TTTTATTATTCATTAAGGGTTAAGTGGGGGGTCTTTAAGATTAAATTCTCTGAATTGTAC
1080 ***************************************.********************
KT719404.1 ATACATGGTTACACGGATATTGTATTCCTGGTCGTATATACTGTTTTCGAACGCAGTGCC
1140OR537693.1
GGAGTGGTAGGAGAAGGGCTGGGTTATGGTATGGCGGGAGGAGTAGTTTACATAGGGGTC 1320OR537692.1
GAAGTGGTAGGAGAAGGGCTGGGTTATGGTATGGCGGGAGGAGTAGTTTACATAGGGGTC 1320 .**********************************************************
Trang 5CCAGCGGTAACGGTGGCGGGGGTGGAGGAGCCAGGGGCGGCGGCGGAGGATCTGGCCAAG 1680OR537692.1
CCAGCGGTAACGGTGGCGGGGGTGGAGGAGCCAGGGGCGGCGGCGGAGGATCTGGCCAAG 1680 ************************** *********************************
Trang 6Biến tính:94-95oC/2-4p; Bắt cặp:66-67oC/30s; Kéo dài:72oC/45s; Kéo dài cuối:72oC/5-10p
BTAP: LÀM PRIMER KHUẾCH ĐẠI ORF1
ORF1(51 995)
ORF1 FOR: 5’ATGCCCAGCAAAAAGAATG3’ (63oC)
ORF1 REV: 5’TCAGTAATTTATTTCATATGGAAATTCAGGGCATGGG3’ (63oC)
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACORF1ATGCCCAGCAAAAAGAATGGAAGAAGCGGACCCCAACCACACAAAAGGTGGGTGTTCACGCTGAATAATCCTTCCGAAGACGAGCGCAAGAAAATACGGGAGCTTCCAATCTCCCTTTTTGATTATTTTATTGTTGGCGAGGAGGGTAATGAGGAAGGACGAACACCCCACCTCCAGGGGTTCGCTAATTTTGTGAAGAAGCAAACATTTAATAAAGTGAAGTGGTATTTCGGTGCCCGCTGCCGCATCGAGAAAGCGAAAGGAACGGATCAGCAGAATAAAGAATATTGCAGTAAAGAAGGCAACTTACTGGTCGAATGTGGAGCTCCTAGATCTCAAGGACAACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGCTGGAGAGCGGGAGTCTGGTGACCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACCAATGTACACGTCATTGTGGGGCCACCTGGGTGTGGCAAAAGTAAATGGGCTGCTAATTTTGCAGACCCGGAAACCACATACTGGAAACCACCTAGAAACAAGTGGTGGGATGGTTACCATGGTGAAGAAGTGGTTGTTATTGATGACTTTTATGGCTGGCTGCCCTGGGATGATCTACTGAGACTGTGTGATCGATATCCATTGACTGTAGAGACTAAAGGTGGAACTGTACCTTTTTTGGCCCGCAGTATTCTGATTACCAGCAATCAGACCCCGTTGGAATGGTACTCCTCAACTGCTGTCCCAGCTGTAGAAGCTCTTTATCGGAGGATTACTTCCTTGGTATTTTGGAAGAATGCTACAGAACAATCCACGGAGGAAGGGGGCCAGTTCGTCACCCTTTCCCCCCCATGCCCTGAATTTCCATATGAAATAAATTACTGAGTCTTTTTTATCACTTCGTAATGGTTTTTATTATTCATTAAGGGTTAAGTGGGGGGTCTTTAAGATTAAATTCTCTGAATTGTACATACATGGTTACACGGATATTGTATTCCTGGTCGTATATACTGTTTTCGAACGCAGTGCCGAGGCCTACGTGGTCTACATTTCCAGTAGTTTGTAGTCTCAGCCACAGCTGATTTCTTTTGTTGTTTGGTTGGAAGTAATCAATAGTGGAATCTAGGACAGGTTCGGGGGTAAAGTAGCGGAAGTGGTAGGAGAAGGGCTGGGTTATGGTATGGCGGGAGGAGTAGTTTACATAGGGGTCATAGGTGAGGGCTGTGGTTTTTGTGAAAAAGTTATCATCTAGAATAACAGCACTGGAGCCCACTCCCCTGTCACCCTGGGTGATCGGGGAGCAGGGCCAGAATTCAACCTTAACCTTTCTTATTCTGTAGTATTCAAAGGGCACAGAGCGGGGGTTTGAGCCCCCTCCTGGGGGAAGAAAGTCGTTAATATTGAATCTCATCATGTCCACCGCCCAGGAGGGCGTTTTGACTGTGGTTCGCTTGATAGTATATCCGAAGGTGCGGGAGAGGCGGGTGTTGAATATGCCATTTTTCCTTCTCCAGCGGTAACGGTGGCGGGGGTGGAGGAGCCAGGGGCGGCGGCGGAGGATCTGGCCAAGATGGCTGCGGGGGCGGTGTCTTCTTCTCCGGTAACGCCTCCTTGGATACGTCATORF2ATCTGAAAACGAAAGAAGTGCGCTGTAAGTATT
REVERSE COMPLEMENT OF ORF2
5’ATGACGTATCCAAGGAGGCGTTACCGGAGAAGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCCTCCACCCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATATTCAACACCCGCCTCTCCCGCACCTTCGGATATACTATCAAGCGAACCACAGTCAAAACGCCCTCCTGGGCGGTGGACATGATGAGATTCAATATTAACGACTTTCTTCCCCCAGGAGGGGGCTCAAACCCCCGCTCTGTGCCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCGATCACCCAGGGTGACAGGGGAGTGGGCTCCAGTGCTGTTATTCTAGATGATAACTTTTTCACAAAAACCACAGCCCTCACCTATGACCCC
Trang 7TATGTAAACTACTCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTTCCGCTACTTTACCCCCGAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGACTACAAACTACTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAACCCTTAA3’ 3’TGGGGGGTGAATTGGGAATT5’=> CP
ORF2 (1734 1033) => ORF2 = 1734-1033-1=702 => Check lại 702 : 3 = 234
Phần 3: Sản phẩm dự kiến sau khi PCR để giới thiệu vị trí cắt của enzyme cắt
vào gene mục tiêu
NheI: GCTAGC
KpnI: GGTACC
atatat: Chỗ bám của enzyme cắt
REVERSE COMPLEMENT OF ORF2
5’atatatgctagcATGTACGTATCCAAGGAGGCGTTACCGGAGAAGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCCTCCACCCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATATTCAACACCCGCCTCTCCCGCACCTTCGGATATACTATCAAGCGAACCACAGTCAAAACGCCCTCCTGGGCGGTGGACATGATGAGATTCAATATTAACGACTTTCTTCCCCCAGGAGGGGGCTCAAACCCCCGCTCTGTGCCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCGATCACCCAGGGTGACAGGGGAGTGGGCTCCAGTGCTGTTATTCTAGATGATAACTTTTTCACAAAAACCACAGCCCTCACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTTCCGCTACTTTACCCCCGAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGACTACAAACTACTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACT
TAACCCTTAAgg taac atatat3’
Thiết kế cặp primer và giới thiệu enzyme cắt vào gen mục tiêu
NHeI-ORF2 FOR: 5’atatatgctagcATGACGTATCCAAGGAGGCG3’(20nt, 67oC)
KpnI-ORF2 REV: 5’atatatggtaacTTAAGGGTTAAGTGGGGGGT3’=> RCP phần không bắt cặp
Trang 8NHeI-ORF2 FOR: 5’atatatgctagcATGACGTATCCAAGGAGGCG3’(20nt, 67oC)
KpnI-ORF2+HA REV: 5’atatatggtaacTTAAGGGTTAAGTGGGGGG3’=> Dùng so sánh
5’atatgttaacTTAAGCGTAATCTGGAACATCGTATGGGTAAGGGTTAAGTGGGGGGT3’
(17nt,66oC)
(Orginal
seq:5’ACCCCCCACTTAACCCTTACCCATACGATGTTCCAGATTACGCTTAA gg taac atatat3’)
Tạo cặp primer giới thiệu HA tag vào ORF2: TACCCATACGATGTTCCAGATTACGCT đặt vào đầu 5’của gene
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTTACGTATCCAAGGAGGCGTTACCGGAGAAGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCCTCCACCCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATATTCAACACCCGCCTCTCCCGCACCTTCGGATATACTATCAAGCGAACCACAGTCAAAACGCCCTCCTGGGCGGTGGACATGATGAGATTCAATATTAACGACTTTCTTCCCCCAGGAGGGGGCTCAAACCCCCGCTCTGTGCCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCGATCACCCAGGGTGACAGGGGAGTGGGCTCCAGTGCTGTTATTCTAGATGATAACTTTTTCACAAAAACCACAGCCCTCACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTTCCGCTACTTTACCCCCGAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGACTACAAACTACTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAACCCTTAAggtaacatatat3’
Biến tính:94-95oC/2-4p; Bắt cặp:66-67oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: ORF2 gene or PCV2 genome
BÀI TẬP: GẮN HA tag hoặc flag tag vào GP5 của PRRSV ở đầu 5’ hoặc 3’
Lấy gene GP5 và GP2 của PRRSV
>AY262352.1:13750-14352 PRRSV HB-2(sh)/2002, complete genome (GP5)
5’ATGTTGGGGAAATGCTTGACCGCGGGCTGTTACTCGCAATTGCTTTCTTTGTGGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCGTCAACGCCAGCAGCAACAGCAGCCCTCATTTTCAGTTGATTTATAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCAGAAAGATTTGACTGGGCAGTGGAGACTTTTGTCATTTTTCCTGTGCTGACTCACATTGTTTCCTATGGAGCACTCACCACCAGCCATTTTCTTGACACGGTTGGTCTAATCACTGTGTCCACCGCCGGGTTCTATCACAGGCGGTATGTCTTGAGTAGTATCTATGCGGTCTGTGCTTTGGCTGCGTTGATTTGCTTCGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACTAGATACACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGCCGCCCGTTATTGTAGAGAAAGGGGGTAAGGTTGAAGTCGAGGGTCACCTGATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGTGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATGGGGTCGTCTCTAG3’
Trang 9KpnI: GGTACC
atatat: Chỗ bám của enzyme cắt
REVERSE COMPLEMENT OF GP5
5’atatatgctagcATGTTGGGGAAATGCTTGACCGCGGGCTGTTACTCGCAATTGCTTTCTTTGTGGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCGTCAACGCCAGCAGCAACAGCAGCCCTCATTTTCAGTTGATTTATAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCAGAAAGATTTGACTGGGCAGTGGAGACTTTTGTCATTTTTCCTGTGCTGACTCACATTGTTTCCTATGGAGCACTCACCACCAGCCATTTTCTTGACACGGTTGGTCTAATCACTGTGTCCACCGCCGGGTTCTATCACAGGCGGTATGTCTTGAGTAGTATCTATGCGGTCTGTGCTTTGGCTGCGTTGATTTGCTTCGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACTAGATACACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGCCGCCCGTTATTGTAGAGAAAGGGGGTAAGGTTGAAGTCGAGGGTCACCTGATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGTGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATGGGGTCGTCTCTAGggtaacatatat3’
Thiết kế cặp primer và giới thiệu enzyme cắt vào gen mục tiêu
NHeI-GP5 FOR: 5’atatatgctagcATGTTGGGGAAATGCTT3’(17nt,58oC)
KpnI-GP5 REV: 5’atatatggtagcCTAGAGACGACCCCAT3’(16nt, 59oC)
Original seq 5’ATGGGGTCGTCTCTAGatatatggtagc3’
Biến tính:94-95oC/2-4p; Bắt cặp:58-59oC/30s; Kéo dài:72oC/45s; Kéo dài cuối:72oC/5-10p
Temple: GP5 gene or PRRVS genome
Tạo cặp primer giới thiệu HA tag vào ORF2: TACCCATACGATGTTCCAGATTACGCT đặt vào đầu
3’của gene
5’atatatgctagcATGTTGGGGAAATGCTTGACCGCGGGCTGTTACTCGCAATTGCTTTCTTTGTGGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCGTCAACGCCAGCAGCAACAGCAGCCCTCATTTTCAGTTGATTTATAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCAGAAAGATTTGACTGGGCAGTGGAGACTTTTGTCATTTTTCCTGTGCTGACTCACATTGTTTCCTATGGAGCACTCACCACCAGCCATTTTCTTGACACGGTTGGTCTAATCACTGTGTCCACCGCCGGGTTCTATCACAGGCGGTATGTCTTGAGTAGTATCTATGCGGTCTGTGCTTTGGCTGCGTTGATTTGCTTCGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACTAGATACACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGCCGCCCGTTATTGTAGAGAAAGGGGGTAAGGTTGAAGTCGAGGGTCACCTGATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGTGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATGGGGTCGTCTCTACCCATACGATGTTCCAGATTACGCTTAGgg t aacatatat3’
NHeI-GP5 FOR: 5’atatatgctagcATGTTGGGGAAATGCTT3’(17nt,58oC)
KpnI-GP5+HA REV:
5’atatatgttaccCTAAGCGTAATCTGGAACATCGTATGGGTAGAGACGACCCCAT3’(13nt,59oC)
Original seq 5’ATGGGGTCGTCTCTACCCATACGATGTTCCAGATTACGCTTAGatatatggtagc3’
Biến tính:94-95oC/2-4p; Bắt cặp:58-59oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: GP5 gene or PRRVS genome
Tạo cặp primer giới thiệu HA tag vào ORF2: TACCCATACGATGTTCCAGATTACGCT đặt vào đầu
5’của gene
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTTTGGGGAAATGCTTGACCGCGGGCTGTTACTCGCAATTGCTTTCTTTGTGGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCGTCAACGCCAGCAGCAACAGCAGCCCTCATTTTCAGTTGATTTATAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCAGAAAGATTTGACTGGGCAGTGGAGACTTTTGTCATTTTTCCTGTGCTGACTCACATTGTTTCCTATGGAGCACTCACCACCAGCCATTTTCTTGACACGGTTGGTCTAATCACTGTGTCCACCGCCGGGTTCTATCACAGGCGGTATGTCTTGAGTAGTATCTATGCGGTCTGTGCTTTGGCTGCGTTGATTTGCTTCGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACTAGATACACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGT
Trang 10CCCTTTAACCAGAGTTTCAGCGGAACAATGGGGTCGTCTCTAGggtaacatatat3’
NHeI-GP5+HA FOR:
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTTTGGGGAAATGCTTGAC3’(17nt,58oC)
KpnI-GP5 REV: 5’atatatggtagcCTAGAGACGACCCCAT3’(16nt, 59oC)
Original seq 5’ATGGGGTCGTCTCTAGatatatggtagc3’
Biến tính:94-95oC/2-4p; Bắt cặp:58-59oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: GP5 gene or PRRVS genome
BÀI TẬP: GẮN HA tag hoặc flag tag vào GP2 của PRRSV ở đầu 5’ hoặc 3’
Lấy gene GP5 và GP2 của PRRSV
>AY262352.1:12038-12808 PRRSV HB-2(sh)/2002, complete genome (GP2)
ATGAAATGGGGTCTATGCAGAGCCTCTTTGACAAAATTGGTCAACTTTTTGTGGATGCTTTCACGGAATTTTTGGTGTCCATTGTTGATATCATCATATTTTTGGCCATTTTGTTTGGCTTCACCATCGCCGGCTGGCTGGTGGTCTTTTGCATCAGACTGGTTTGCTCCGCGATATTCCGTGCGCGCCCTACCGTTCACCCTGAGCAATTACAGAAGATCCTATGAGGCCTTTCTTTCTCAATGCCAGGTGGACATTCCCACCTGGGGCGTTAAACACCCTTTGGGAATACTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCATCATGGAAAAAGCAGGGCAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCTACACTGTCTCGCATTAGTAATTTGGATGTGGTGGCTCATTTTCAACACCTTGCCGCCATTGAGGCCGAGACCTACAAATACTTGGCTTCCCGGCTGCCCATGCTTCACAACCTGCGCATGACAGGGTCAAATGTAACCATAGTGTATAATAGTACCTTGAATCAGGTATTTGCTATTTTCCCAACCTCTGGTTCCCGACCAAGGCTCCATGATTCTCAGCAATGGCTAATAGCTGTACATTCCTCCATATTTTCCTCTGTTGTGGCTTCTTGTACTCTTTTTGTTGTGCTGTGGTTGCGAATTCCAATGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCTTTTGAACTCACGGTGA
Làm primer khuếch đại GP2
Thiết kế cặp primer và giới thiệu enzyme cắt vào gen mục tiêu
NHeI-GP2 FOR: 5’atatatgctagcATGAAATGGGGTCTATGCA3’(20nt,62oC)
Trang 11KpnI-GP2 REV: 5’atatatgttaccTCACCGTGAGTTCAAAAGAA3’(20nt,61oC)
Original seq 5’TTCTTTTGAACTCACGGTGAggtaacatatat3’
Biến tính:94-95oC/2-4p; Bắt cặp:62-61oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: GP5 gene or PRRVS genome
Tạo cặp primer giới thiệu HA tag vào ORF2: TACCCATACGATGTTCCAGATTACGCT đặt vào đầu
3’của gene
5’atatatgctagcATGAAATGGGGTCTATGCAGAGCCTCTTTGACAAAATTGGTCAACTTTTTGTGGATGCTTTCACGGAATTTTTGGTGTCCATTGTTGATATCATCATATTTTTGGCCATTTTGTTTGGCTTCACCATCGCCGGCTGGCTGGTGGTCTTTTGCATCAGACTGGTTTGCTCCGCGATATTCCGTGCGCGCCCTACCGTTCACCCTGAGCAATTACAGAAGATCCTATGAGGCCTTTCTTTCTCAATGCCAGGTGGACATTCCCACCTGGGGCGTTAAACACCCTTTGGGAATACTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCATCATGGAAAAAGCAGGGCAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCTACACTGTCTCGCATTAGTAATTTGGATGTGGTGGCTCATTTTCAACACCTTGCCGCCATTGAGGCCGAGACCTACAAATACTTGGCTTCCCGGCTGCCCATGCTTCACAACCTGCGCATGACAGGGTCAAATGTAACCATAGTGTATAATAGTACCTTGAATCAGGTATTTGCTATTTTCCCAACCTCTGGTTCCCGACCAAGGCTCCATGATTCTCAGCAATGGCTAATAGCTGTACATTCCTCCATATTTTCCTCTGTTGTGGCTTCTTGTACTCTTTTTGTTGTGCTGTGGTTGCGAATTCCAATGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCTTTTGAACTCACGGTACCCATACG
NHeI-GP2 FOR: 5’atatatgctagcATGAAATGGGGTCTATGCA3’(20nt,62oC)
KpnI-GP2+HA REV:
5’TTCTTTTGAACTCACGGTACCCATACGATGTTCCAGATTACGCTTGAggtaacatatat3’(17nt,61oC)Original seq 5’TTCTTTTGAACTCACGGTACCCATACGATGTTCCAGATTACGCTTGAggtaacatatat3’Biến tính:94-95oC/2-4p; Bắt cặp:61-62oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: GP2 gene or PRRVS genome
Tạo cặp primer giới thiệu HA tag vào ORF2: TACCCATACGATGTTCCAGATTACGCT đặt vào đầu
5’của gene
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTAAATGGGGTCTATGCAGAGCCTCTTTGACAAAATTGGTCAACTTTTTGTGGATGCTTTCACGGAATTTTTGGTGTCCATTGTTGATATCATCATATTTTTGGCCATTTTGTTTGGCTTCACCATCGCCGGCTGGCTGGTGGTCTTTTGCATCAGACTGGTTTGCTCCGCGATATTCCGTGCGCGCCCTACCGTTCACCCTGAGCAATTACAGAAGATCCTATGAGGCCTTTCTTTCTCAATGCCAGGTGGACATTCCCACCTGGGGCGTTAAACACCCTTTGGGAATACTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCATCATGGAAAAAGCAGGGCAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCTACACTGTCTCGCATTAGTAATTTGGATGTGGTGGCTCATTTTCAACACCTTGCCGCCATTGAGGCCGAGACCTACAAATACTTGGCTTCCCGGCTGCCCATGCTTCACAACCTGCGCATGACAGGGTCAAATGTAACCATAGTGTATAATAGTACCTTGAATCAGGTATTTGCTATTTTCCCAACCTCTGGTTCCCGACCAAGGCTCCATGATTCTCAGCAATGGCTAATAGCTGTACATTCCTCCATATTTTCCTCTGTTGTGGCTTCTTGTACTCTTTTTGTTGTGCTGTGGTTGCGAATTCCAATGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCTTTTGAACTCACGGTGAggtaacatatat3’
NHeI-GP2+HA FOR:
5’atatatgctagcATGTACCCATACGATGTTCCAGATTACGCTAAATGGGGTCTATGCAGAG3’(19nt,61oC)KpnI-GP2 REV: 5’atatatgttaccTCACCGTGAGTTCAAAAGAA3’(20nt,61oC)
Original seq 5’TTCTTTTGAACTCACGGTGAggtaacatatat3’
Biến tính:94-95oC/2-4p; Bắt cặp:61oC/30s; Kéo dài: 72oC/45s; Kéo dài cuối:72oC/5-10p;
Temple: GP5 gene or PRRVS genome
Hãy tiếp hợp GP2 với GP5 để tạo gene tiếp hợp (ATG…Stop) với GP5 nằm ở 5’ của GP2 Sẵn phẩm mục tiêu
Trang 12ATGAAATGGGTGCTCTACAAAGCCTTTTTGAAAAAATTGGCCAACTTTTTGTGGATGCCTTCACGGAGTTCTTGGTGTCTATTGTTGACATTGTCATATTTCTGGCCATCTTGTTTGGCTTTACAGTCGCCGGTTGGCTGGTTGTCTTTTGCATCAGATTGGTTTGCTCCACGATACTCCGTGCGCGCTCTGCCGTTCACTCTGAGCAGTTACAGAAGATCCTATGAGGTTTTCCTTCCCCTGTGCAAGACGGATGTCCCCGTCTGGGGAACCAAGCACCCCCTAGGAATGTTCTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCACCATGGAAAATGCAGGACAGGCTGCCTGGAAACAGGTGGTGAGTGAGGCTACTCTGTCTCGCATTAGCAGCTTGGATGTGGTGGCTCATTTCCAGCACCTCGCCGCCATTGAAGCCGAGACCTGTAAATACTTGGCTTCCCGGCTGCCCATGCTGCACAACCTGCGCATGACTGGGTCGAATGTAACCATAGTGTATAACAGCACTTCGGATCAGGTGTCTGCTATTTTCCCAACCCCCGGCTCCCGGCCAAAACTTCATGATTTTCAGCAATGGCTGATAGCTGTACATTCCTCTATATTCTCTTCTGTTGCGGCTTCTTGCACTCTCTTTGTTGTGTTGTGGTTGCGAATTCCAACGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCCTTGTGA
GP5
ATGTTGGGGAAATGCTTGACCGCGGGCTGCTGCTCGCAATTGCCTTTTTTGTGGTGTATCGTGCCGTTCTGTCTTGTTGCGCTCGTCAACGCCAGCAACGGCAGCAGCTCCCATTTACAGTTGATTTATAACCTGACGATATGCGAGCTGAATGGCACAGATTGGCTAAATAAAAGTTTTGATTGGGCGGTGGAGACTTTTGTCATCTTTCCTGTGTTGACTCACATTGTCTCCTATGGCGCCCTTACCACTAGTCATTTCCTTGACATGGTCGGCCTGATCACCGTGTCTACCGCCGGGTATTACCACGGACGGTATGTCTTGAGTAGCATTTACGCCGTCTGCGCCCTGGCTGCGTTAATTTGCTTCGTCATCAGGCTAACAAAAAATTGTATGTCCTGGCGTTACTCATGCACCAGATACACTAATTTTCTTCTGGACACCAAGGGCAAACTCTATCGTTGGCGGTCTCCTGTCATCATAGAAAAAGGGGGTAAAGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTGTAACCAAAGTTTCAGCGGAACAATGGGGTCGTCCTTAG
PCR1: Ưu tiên nhân gene ngắn hơn lên trước Nhân GP5 lên trước:
atatatgctagcATGTTGGGGAAATGCTTGACCGCGGGCTGCTGCTCGCAATTGCCTTTTTTGTGGTGTATCG
TGCCGTTCTGTCTTGTTGCGCTCGTCAACGCCAGCAACGGCAGCAGCTCCCATTTACAGTTGATTTATAACCTGACGATATGCGAGCTGAATGGCACAGATTGGCTAAATAAAAGTTTTGATTGGGCGGTGGAGACTTTTGTCATCTTTCCTGTGTTGACTCACATTGTCTCCTATGGCGCCCTTACCACTAGTCATTTCCTTGACATGGTCGGCCTGATCACCGTGTCTACCGCCGGGTATTACCACGGACGGTATGTCTTGAGTAGCATTTACGCCGTCTGCGCCCTGGCTGCGTTAATTTGCTTCGTCATCAGGCTAACAAAAAATTGTATGTCCTGGCGTTACTCATGCACCAGATACACTAATTTTCTTCTGGACACCAAGGGCAAACTCTATCGTTGGCGGTCTCCTGTCATCATAGAAAAAGGGGGTAAAGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTGTAACCAAAGTTTCAGCGGAACAATGGGGTCGTCCT///
ATGAAATGGGTGCTCTACAAAGCCTTTTTGAAAAAATTGGCCAACTTTTTGTGGATGCCTTCACGGAGTTCTTGGTGTCTATTGTTGACATTGTCATATTTCTGGCCATCTTGTTTGGCTTTACAGTCGCCGGTTGGCTGGTTGTCTTTTGCATCAGATTGGTTTGCTCCACGATACTCCGTGCGCGCTCTGCCGTTCACTCTGAGCAGTTACAGAAGATCCTATGAGGTTTTCCTTCCCCTGTGCAAGACGGATGTCCCCGTCTGGGGAACCAAGCACCCCCTAGGAATGTTCTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCACCATGGAAAATGCAGGACAGGCTGCCTGGAAACAGGTGGTGAGTGAGGCTACTCTGTCTCGCATTAGCAGCTTGGATGTGGTGGCTCATTTCCAGCACCTCGCCGCCATTGAAGCCGAGACCTGTAAATACTTGGCTTCCCGGCTGCCCATGCTGCACAACCTGCGCATGACTGGGTCGAATGTAACCATAGTGTATAACAGCACTTCGGATCAGGTGTCTGCTATTTTCCCAACCCCCGGCTCCCGGCCAAAACTTCATGATTTTCAGCAATGGCTGATAGCTGTACATTCCTCTATATTCTCTTCTGTTGCGGCTTCTTGCACTCTCTTTGTTGTGTTGTGGTTGCGAATTCCAACGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCCTTGTGAgttaccatatat
Sẵn phẩm mục tiêu: Nhel (gctagc) và Kpnl (gttaac)
Trang 13Chu kỳ nhiệt: 94oC/3p;35 chu kỹ với chu kì nhiệt:63oC/30s;72oc/45s;72oC/5p
PCR2:
GP2-GP5 For: 5’atatatgctagcATGTTGGGGAAATGCTTGACTTGTAGAGCACCCATTTCAT3’ (20nt;63oC)Sản phẩm của PCR1
GP2-Kpn1 REV: atatatgttaacTCACAAGGAGCTCGAAG (17nt,61oC)
(Original Seq: CTTCGAGCTCCTTGTGAgttaccatatat
Template PCR2: GP2
Chu kỳ nhiệt: 94oC/3p;35 chu kỹ với chu kì nhiệt:63oC/30s;72oc/45s;72oC/5p
Giả sử: Muốn nhân gene đứng sau ở PCR1
atatatgctagcATGTTGGGGAAATGCTTGACCGCGGGCTGCTGCTCGCAATTGCCTTTTTTGTGGTGTATCG
TGCCGTTCTGTCTTGTTGCGCTCGTCAACGCCAGCAACGGCAGCAGCTCCCATTTACAGTTGATTTATAACCTGACGATATGCGAGCTGAATGGCACAGATTGGCTAAATAAAAGTTTTGATTGGGCGGTGGAGACTTTTGTCATCTTTCCTGTGTTGACTCACATTGTCTCCTATGGCGCCCTTACCACTAGTCATTTCCTTGACATGGTCGGCCTGATCACCGTGTCTACCGCCGGGTATTACCACGGACGGTATGTCTTGAGTAGCATTTACGCCGTCTGCGCCCTGGCTGCGTTAATTTGCTTCGTCATCAGGCTAACAAAAAATTGTATGTCCTGGCGTTACTCATGCACCAGATACACTAATTTTCTTCTGGACACCAAGGGCAAACTCTATCGTTGGCGGTCTCCTGTCATCATAGAAAAAGGGGGTAAAGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTGTAACCAAAGTTTCAGCGGAACAATGGGGTCGTCCT///
ATGAAATGGGTGCTCTACAAAGCCTTTTTGAAAAAATTGGCCAACTTTTTGTGGATGCCTTCACGGAGTTCTTGGTGTCTATTGTTGACATTGTCATATTTCTGGCCATCTTGTTTGGCTTTACAGTCGCCGGTTGGCTGGTTGTCTTTTGCATCAGATTGGTTTGCTCCACGATACTCCGTGCGCGCTCTGCCGTTCACTCTGAGCAGTTACAGAAGATCCTATGAGGTTTTCCTTCCCCTGTGCAAGACGGATGTCCCCGTCTGGGGAACCAAGCACCCCCTAGGAATGTTCTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATGTACCGCACCATGGAAAATGCAGGACAGGCTGCCTGGAAACAGGTGGTGAGTGAGGCTACTCTGTCTCGCATTAGCAGCTTGGATGTGGTGGCTCATTTCCAGCACCTCGCCGCCATTGAAGCCGAGACCTGTAAATACTTGGCTTCCCGGCTGCCCATGCTGCACAACCTGCGCATGACTGGGTCGAATGTAACCATAGTGTATAACAGCACTTCGGATCAGGTGTCTGCTATTTTCCCAACCCCCGGCTCCCGGCCAAAACTTCATGATTTTCAGCAATGGCTGATAGCTGTACATTCCTCTATATTCTCTTCTGTTGCGGCTTCTTGCACTCTCTTTGTTGTGTTGTGGTTGCGAATTCCAACGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCCTTGTGAgttaccatatat
Sẵn phẩm mục tiêu: Nhel (gctagc) và Kpnl (gttaac)
Chu kỳ nhiệt: 94oC/3p;35 chu kỹ với chu kì nhiệt:61oC/30s;72oc/45s;72oC/5p
PCR2: Sợi trên hay dưới của sản phẩm PCR1 sẽ tham gia vào phản ứng PCR2
GP5-Nhel For: 5’atatatgctagcATGTTGGGGAAATGCTTGAC3’ (20nt, 63oC)
GP2-GP5 REV:atatatggtaacTCACAAGGAGCTCGAAGAGGACGACCCCATTGTT
(Original Seq: (17nt,63oC)AACAATGGGGTCGTCCTCTTCGAGCTCCTTGTGAgttaccatatat(20nt,63oC))
Hỏi đây là sợi trên hay dưới của sản phẩm PCR1: Sợi dưới
Template PCR2: GP5
Chu kỳ nhiệt: 94oC/3p;35 chu kỹ với chu kì nhiệt:63oC/30s;72oc/45s;72oC/5p
GP5 CỦA VIRUS PRRSV TẠO ĐỘT BIẾN Ở CÁC VỊ TRÍ AA SỐ 50,51,55 THÀNH ALANIN (L50A,N51A và W55A)
153:3=51 Ra 55 là 4x3= 12
Trang 14MLGKCLTAGCCSQLPFLWCIVPFCLVALVNASNGSSSHLQLIYNLTICELNGTDWLNKSFDWAVETFVIFPVLTHIVSYGALTTSHFLDMVGLITVSTAGYYHGRYVLSSIYAVCALAALICFVIRLTKNCMSWRYSCTRYTNFLLDTKGKLYRWRSPVIIEKGGKVEVEGHLIDLKRVVLDGSAATPVTKVSAEQWGRP-
CCCTGTAACCAAAGTTTCAGCGGAACAATGGGGTCGTCCTTAGgttaccatatat
MLGKCLTAGCCSQLPFLWCIVPFCLVALVNASNGSSSHLQLIYNLTICEAAGTDA
PCR1:Nhân vùng gene ngắn hơn lên trước
For: atatatgctagcATGTTGGGGAAATGCTTGA (19nt,62oC)
Trang 15FOR: atatatgctagcATGTTGGGGAAATGCTTGACGCATATCGTCAGGTTAT (19nt,62oC)
Sản phẩm PCR1 cung cấp sợi ở trên có chức năng là primer forward
REV: atatatggtaacCTAAGGACGACCCCATTG (18nt,62oC)
(original seq: CAATGGGGTCGTCCTTAGgttaccatatat)
Temple của PCR2:
Chu kỳ nhiệt: 94oC/3p;35 chu kỹ với chu kì nhiệt:62oC/30s;72oc/45s;72oC/5p
Lấy gene GP3 và N (ORF7) của PRRSV và tiếp hợp 2 gene này với GP3 ở đầu 5' của ORF7.
GP3
ATGGCTGATAGCTGTACATTCCTCTATATTCTCTTCTGTTGCGGCTTCTTGCACTCTCTTTGTTGTGTTGTGGTTGCGAATTCCAACGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCCTTGTGAATTACACGGTGTGCCAGCCTTGCCTTACTCGACAAGCAGCCGCTCAGATTTATGAACCTGGCAGATCCCTTTGGTGCAGGATTGGGCATGATCGATGTGAGGAGAACGACCATGACGAGCTAGGATTTGTGGTACCGCCTGGTCTTTCCAGCGAAGGCCATTTAACCAGTGCTTATGCCTGGTTGGCGTTCTTGTCCTTCAGTTATACAGCCCAGTTTCATCCCGAGATATTCGGAATCGGGAACGTGAGCACCGTTTATGTTGACAAACCCCATCAACTCATTTGTGCCGTTCATGATGGGCAGAATGCCACCTTGTCCCGCTACGATAACATCACAGCCGTGTATCAGACCTACTATCAACACCAAGTTGATGGCGGCAATTGGTTTCACCTAGAATGGCTGCGTCCTTTCTTTTCCTCTTGGTTGGTTTTGAACGTTTCATGGTTTCTCAGGCGTTCGCCTGTAAACCGTGTTTCAGTTCGAGTCTTTCAGATATCAAGATCAATACCACCAGCGCAGCAAGTTCTGTTGTCCTCCAAGACATCAGCTGTCTTAGGCATAGCAACCCGTCCTCTGCGACGTTTCGCAAAATCCCTCAGTGCCGTACGGCGATAG
ORF7
ATGCCAAATAACAACGGCAGACAGCAAAAGAAAAAGAAGGGGGATGGCCAGCCAGTCGATCAGCTTTGCCAGATGTTGGGCAAGATTATCGCCCAACAGAGCCAGTCCAGAGGTAGGGGACCGGGGAAGAAGAATAATAATAGAAACCCGGAAAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGACATCACTTTATCCCCAGTGAGCGACAATTGTGTCTGTCGTCAATCCGGACTGCCTTTAATCAAGGCGCTGGAACTTGTACCCTGTCAGACTCAGGGAGAATAAGTTACACTGTGGAGTTCAGTCTGCCTACTCGTCATACTGTGCGCTTGATTCGCGCCACAGCGTCACCCTCAGCATGA
Sẵn phẩm mục tiêu: Nhel (gctagc) và Kpnl (gttaac)
atatatgctagcATGGCTGATAGCTGTACATTCCTCTATATTCTCTTCTGTTGCGGCTTCTTGCACTCTCTTTGT
TGTGTTGTGGTTGCGAATTCCAACGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCCTTGTGAATTACACGGTGTGCCAGCCTTGCCTTACTCGACAAGCAGCCGCTCAGATTTATGAACCTGGCAGATCCCTTTGGTGCAGGATTGGGCATGATCGATGTGAGGAGAACGACCATGACGAGCTAGGATTTGTGGTACCGCCTGGTCTTTCCAGCGAAGGCCATTTAACCAGTGCTTATGCCTGGTTGGCGTTCTTGTCCTTCAGTTATACAGCCCAGTTTCATCCCGAGATATTCGGAATCGGGAACGTGAGCACCGTTTATGTTGACAAACCCCATCAACTCATTTGTGCCGTTCATGATGGGCAGAATGCCACCTTGTCCCGCTACGATAACATCACAGCCGTGTATCAGACCTACTATCAACACCAAGTTGATGGCGGCAATTGGTTTCACCTAGAATGGCTGCGTCCTTTCTTTTCCTCTTGGTTGGTTTTGAACGTTTCATGGTTTCTCAGGCGTTCGCCTGTAAACCGTGTTTCAGTTCGAGTCTTTCAGATATCAAGATCAATACCACCAGCGCAGCAAGTTCTGTTGTCCTCCAAGACATCAGCTGTCTTAGGCATAGCAACCCGTCCTCTGCGACGTTTCGCAAAATCCCTCAGTGCCGTACGGCGA///
ATGCCAAATAACAACGGCAGACAGCAAAAGAAAAAGAAGGGGGATGGCCAGCCAGTCGATCAGCTTTGCCAGATGTTGGGCAAGATTATCGCCCAACAGAGCCAGTCCAGAGGTAGGGGACCGGGGAAGAAGAATAATAATAGAAACCCGGAAAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGACATCACTTTATCCCCAGTGAGCGACAATTGTGTCTGTCGTCAATCCGGACTGCCTTTAATCAAGGCGCTGGAACTTGTACCCTGTCAGACTCAGGGAGAATAAGTTACACTGTGGAGTTCAGTCTGCCTACTCGTCATACTGTGCGCTTGATTCGCGCCACA
GCGTCACCCTCAGCATGAgttaccatatat
Trang 16GP3-Nhel For: 5’atatatgctagcATGGCTGATAGCTGTACATTCC3’ (22nt;64oC)
GP3-ORF7 Rev: 5’ CTGCCGTTGTTATTTGGCATTCGCCGTACGGCACT 3’
(Original seq: 15nt, 67oC):AGTGCCGTACGGCGAATGCCAAATAACAACGGCAG(20nt,64oC)
Chu kỳ nhiệt: 94 o C/3p;35 chu kỹ với chu kì nhiệt:65 o C/30s;72 o c/45s;72 o C/5p
Bài tập kiểm tra việc học: PCV2, PRRSV, dùng gene nào cũng được và làm bài tập cho các mục tiêu như trong file powerpoint
1 Primer cho PCR kiểm tra có – không của PRRSV
ON584463.1 - 0
AY150312.1 ATGACGTATAGGTGTTGGCTCTACGCCACGACATTTGTATTGTCAGGAGCTGTGACCATT
60
ON584463.1 -CTTGCCGCACAGAAACACCCTTCTGTGACGGCCTCCTTCAGGGAGC 46AY150312.1
GGCACAGCCCAAAACTTGCTGCACGGGAACACCCTCTCGTGACAGCCCTCTTCAGGGGGA 120 ***** ****.*.******** *****.*** ********.*
ON584463.1 TTAGGGGTC-GCCCCTAGCACCTTGTTTCTGGAGTTGCACTGCTTTACGGTCTCTCCACC
105AY150312.1 TTAGGGGTCTGTCCCTAACACCTTGCTTCCGGAGTTGCACTGCTTTACGGTCTCTCCACC
180 ********* * *****.******* *** ******************************
ON584463.1 CCTTTAACCATGTCTGGGATTCTTGATCGGTGCACGTGCACCCCCAATGCCAGGGTGTTT
165AY150312.1 CTTTTAACCATGTCTGGGATACTAGATCGGTGCACGTGTACCCCCAATGCCAGGGTGTTT
240
* ******************:**:************** *********************
ON584463.1
GTGGCAGAGGGCCAAGTCTACTGCACACGATGTCTCAGTGCACGGTCCCTCCTCCCCTTG 225AY150312.1
GTGGCGGAGGGCCAGGTCTACTGCACACGATGTCTCAGTGCACGGTCTCTCCTTCCTCTG 300 *****.********.******************************** ***** ** **
ON584463.1
AATCTCCAAGCCCCTGAGCTTGGAGTGCTGGGCTTGTTTTATAGGCCTGAAGAGCCACTC 285AY150312.1 AATCTCCAAGTTCCTGAGCTTGGGGTGCTGGGCCTATTTTATAGGCCCGAAGAGCCACTC
360 ********** ***********.********* *.*********** ************
Trang 17ON584463.1
CGGTGGACGTTGCCACGCGCATTCCCCACTGTTGAGTGCTCCCCCGCTGGGGCCTGTTGG 345AY150312.1
CGGTGGACGTTGCCACGTGCATTCCCCACTGTCGAGTGTTCCCCCACCGGGGCCTGCTGG 420 ***************** ************** ***** ******.* ******** ***
ON584463.1
CTTTCCACGATTTTCCCAATTGCACGGATGACCAGTGGGAACCTGAACTTCCAGCAGAGA 405AY150312.1 CTTTCTGCGATCTTTCCGATTGCACGAATGACCAGTGGTAACCTGAACTTTCAACAAAGA
480 ***** **** ** **.********.*********** *********** **.**.***
ON584463.1
ATGGTACGGGTTGCCGCTGAGATTTACAGAGCTGGCCAACTCACCCCCACAGTCTTGAAA 465AY150312.1
ATGGTGCGGGTCGCAGGTGAAATCTACAGAGCCGGCCAACTCACCCCTACAGTTCTGAAG 540 *****.***** **.* ***.** ******** ************** ***** ****
ON584463.1
AACTTGCAAGTCTATGAACGGGGTTGCCGCTGGTACCCCATCGTCGGACCAGTTCCTGGA 525AY150312.1
ACTATACAAGTTTATGAACGGGGTTGTCGCTGGTACCCCATTGTCGGGCCCGTCCCTGGG 600 * :*.***** ************** ************** *****.**.** *****
ON584463.1 GTTGCCGTTTTCGCCAACTCCCTACATGTGAGTGACAAACCTTTTCCAGGTGCCACTCAC
585AY150312.1 GTGGGCGTCTACGCCAACTCCCTACATGTGAGTGACAAACCTTTCCCGGGAGCAACTCAT
660 ** * *** *:********************************* **.**:**.*****
ON584463.1 GTGCTAACCAACCTGCCGCTTCCGCAGAGACCTAAGCCTGAAGATTTTTGCCCCTTTGAG
645AY150312.1 GTGTTAACCAACTTACCGCTCCCGCAGAGGCCCAAGCCTGAGGACTTTTGCCCTTTTGAG
720 *** ******** *.***** ********.** ********.** ******** ******
ON584463.1
TGTGCTATGGCTGCCGTCTATGATATCGGTCGTGACGCTGTCATGTTCGTGGCCGGAAGG 705AY150312.1
TGTGCTATGGCTGACGTCTATGACATTGGTCGTGGCGCCGTCATGTATGTGGCCGGAGGA 780 *************.********* ** *******.*** *******: *********.*
ON584463.1
AAAGTCTCCTGGGCTCCGCGTGGCGGGGACAAAGGAAGATTTGAAATCACTCCTGAGGGA 765AY150312.1
AAAGTCTCCTGGGCCCCTCGTGGTGGGGATGAAGTGAAATTTGAACCTGTCCCCAAGGAG 840 ************** ** ***** ***** *** *.******* ** ***
ON584463.1
CTAAGGTTGATTGCGGAGCGACTTTACACCTCCTTCCCGCCTCACCACGTGGTGGATATG 825AY150312.1 TTGAAGTTGGTTGCGAACCGACTCCACACCTCCTTCCCGCCCCATCACGTAGTGGACATG
900 *.*.****.*****.* ***** **************** ** *****.***** ***
Trang 18ON584463.1
TCGGAGTTTACCTTTACGGCCCCTGAGTGTGGAGCCTCCATGCGGGTCGAGCGCCAGTAT 885AY150312.1 TCTAAGTTTACCTTCATGACCCCTGGGAGTGGTGTCTCCATGAGGGTCGAGTATCAATAC
960 ** ********** * *.******.*:****:* *******.******** **.**
ON584463.1
GGTTGCCTCCCCGCTGGCACTGTCCCTGATGGCAACTGCTGGTGGAGCTTGTTTGGCTCA 945AY150312.1
GGCTGCCTCCCCGCTGACACTGTCCCTGAAGGAAACTGCTGGTGGCGCTTGTTTGACTTG 1020 ** *************.************:**.************.*********.**
ON584463.1
CTTCCATTGGAAGTTCAGTACAAAGAAATTCGCCGCGCCAACCAATTGGGCTATCAGACC 1005AY150312.1 CTCCCACCGGAAGTTCAGAATAAAGAAATTCGTCATGCTAACCAATTTGGCTATCAAACC
1080 ** *** **********:* *********** * ** ******** ********.***
ON584463.1
AAGCATGGTGTCGCTGGTAAGTACCTGCAGCGAAGGATGCAAGTTAATGGAATCCGGGCA 1065AY150312.1
AAGCATGGTGTCCCTGGTAAGTACCTACAGCGGAGGCTGCAAGTTAACGGTCTTCGAGCA 1140 ************ *************.*****.***.********** **:.* **.***
ON584463.1
GTGGCTGACGCGAGTGGACCCATCGTTGTACAGTACTTCTGCGTTAAAGAAAGCTGGATT 1125AY150312.1 GTGACCGACACACATGGACCTATCGTCATACAGTACTTCTCTGTTAAGGAGAGTTGGATC
1200 ***.* ***.* ****** ***** ************ *****.**.** *****
ON584463.1
CGCCATTTGAGATTGGTAGAGGAACCCAGCCTCCCCGGGTTTGAGGACCTTATCAGAATA 1185AY150312.1
CGCCACCTGAAGCCGGTGGAAGAACCCAGCCTCCCCGGGTTTGAGGATCTCCTCAGAATC 1260 ***** *** ***.**.************************** ** *******
ON584463.1
AGAGTAGAACCCAATACGTCACCATTGGTTGGTGAGGATGAGAAAATATTCCGGTTTGGC 1245AY150312.1
AGGGTTGAGCCCAATACGTCTCCACTGGCTGGAAAGAATGAGAAGATTTTCCGGTTTGGC 1320 **.**:**.***********:*** *** ***:.**.*******.**:************
ON584463.1
GCCCACAAATGGTACGGTGCTGGAAAGAGAGCAAGGAAAGCGCGCTCTAGTGTGGCTACC 1305AY150312.1
AGTCATAAGTGGTACGGTGCTGGAAAGAGAGCAAGGAAAGCACGCTCTGGTGCGACTACC 1380 ** **.********************************.******.*** *.*****
ON584463.1
ACAGCCACTCGCCATGTCTCGCCCGTCCGTGAGACCCAACAAGCCAAAAAACACGATGAC 1365AY150312.1
ATGGTCGCTCATCGCGCTTCGTCCGCTCATGAAACCCGGCAGGCCACGAAGCACGAGGGT 1440
* * *.*** * * *** *** *.***.**** **.**** **.***** *
Trang 19ON584463.1
GTCAGTGCCGACGCGACTGGGCGGTTCGAGCACTACTCTCCGCCAACCGACGGGAATTGT 1425AY150312.1
GCCGGTGCTAACAAGGCTGAGCATCTCAAGCTCTACTCTCCGCCTGCCGAAGGGAACTGT 1500
* *.**** ** *.***.** **.***:************:.****.***** ***
ON584463.1 GGTTGGCATTGCATTTCCGCCATCGTTAACCATGTTGTGAATTCCAAACTTGAAACTACC
1485AY150312.1 GGTTGGCACTGCATTTCCGCCATCGTCAACCGGATGGTGAATTCCAACTTTGAGACCACC
1560 ******** ***************** **** .* *********** ****.** ***
ON584463.1
CTTCCCGAGAGGGTGAGACCTTCAGATGATTGGGCCACTGACGAGGACCTCGTGACTGCT 1545AY150312.1
CTTCCTGAAAGAGTAAGGCCTCCAGATGACTGGGCCACCGACGAGGATCTTGTGAACACC 1620 ***** **.**.**.**.*** ******* ******** ******** ** **** .*
ON584463.1
ATCCAAGTCCTCAGGCTCCCTGCGGCCTTAGACAGGAACGGTGCTTGTGCTGGCGCCAAG 1605AY150312.1
ATCCAAATCCTCAGGCTCCCTGCGGCCTTGGACAGGAACGGCGCTTGCGGTGGCGCCAAG 1680 ******.**********************.*********** ***** * **********
ON584463.1 TACATTCTCAAGTTGGAAGGCGAGCATTGGACTGTCTCGGTCATCCCTGGCATGTCTCCC
1665AY150312.1 TACGTGCTTAAACTGGAGGGTGAGCATTGGACTGTCTCTGTGAACCCTGGGATGTCCCCT
1740 ***.* ** ** ****.** ***************** ** *:****** ***** **
ON584463.1
TCTCTGCTTCCTCTTGAGTGCGCTCAGGGTTGCTGTGAGCACAAGGGTGGCCTTGGCTCC 1725AY150312.1 TCTTTGCTCCCCCTTGAATGTGTTCAGGGCTGTTGTGAGCATAAGGGCGGTCTTGGTTCC
1800 *** **** ** *****.** * ****** ** ******** ***** ** ***** ***
ON584463.1
CCAAACGCGGTCGAAGTTTCTGGGTTTGACCCCGCTTGCCTTGACTGGTTGGCCAAAGTG 1785AY150312.1 CCGGATGCGGTCGAAGTTTCCGGATTTGATCCTGCCTGTCTTGACCGACTGTTACAGGTA
1860 ** * ************** **.***** ** ** ** ****** * ** *.**
ON584463.1
ATGCATCTGCCTAGTAGTGTTGTCCCGGCCGCCTTGGCCGAGATGTCCGGCAATTTTGGT 1845AY150312.1 ATGCACTTGCCTAGCAGTACCATCCCAGCCGCTCTGGCCGAATTGTCCGACGACTCCAAT
1920 ***** ******* *** .****.***** *******.:******.*.* * *
ON584463.1 CGCTCAACCCCTGCGGCTCCTGTTGTATGGACTGTTTCACAATTCTTTGCCCGCCACAGC
1905AY150312.1 CGTCCGGTTTCCCCGGCCGCTGCTACGTGGACTGTTTCGCAATCCTATGCTCGTCATAGA
1980 ** * * **** *** * .***********.**** **:*** ** ** **
Trang 20ON584463.1
GGAGGGGAGCACTCTGACCAAGTGTGTTTGGAGAAAATTGTTAGCCTTTGTCAACTGCTT 1965AY150312.1 GGAGGGAATCATCATGACCAGGTGTGCTTAGGGAAAATCATCAGCCTTTGTCAAGTTATT
2040 ******.* ** ******.***** **.*.****** * ************ * **
ON584463.1
GAGAGCTGCTGCTGCTCTCAGAATAAAGCCAACCCGGTTACCCCGGATGAAGTGAGGAGC 2025AY150312.1
GAGGATTGCTGCTGCCATCAGAATAAAACTAACCGGGCCACTCCGGAAGAGGTCGCGGCA 2100 *** ********* **********.* **** ** ** *****:**.** *
ON584463.1
AAAATTGACCAGTACCTTCGCGGTGCAGCAAATCTTGAAGAATGCTTGGCTAGGCTTGAG 2085AY150312.1 AAAATTGATCAGTACCTCCGTGGCGCAACAAGTCTTGAAGAATGCTTGGCCAAACTTGAG
2160 ******** ******** ** ** ***.***.****************** * ******
ON584463.1
AAAGCTCGCCCGCCAAGCGTGTTGGACACCTCCTTTGATTGGGACGTTGTGCTTCCTGGA 2145AY150312.1
AGAGTTTCCCCGCCGGGCGCTGCGGACACCTCCTTTGATTGGAATGTTGTGCTTCCTGGG 2220 *.** * ****** *** *******************.* **************
ON584463.1
GTTGGAGTGGCAGCTCAGGCAACGGAACTGCCCCCCATTAGTCAGTGTCACACCCTTGTC 2205AY150312.1
GTTGAGGCGGCGCATCAGACAACCGAACAACTTCACGTCAACCCGTGCCGCACCCTGGTT 2280 **** * *** .****.**** ****:.* *.*.* * *.*** *.****** **
ON584463.1 ACTGTCGTGGCCCAAAAGCCTTTG - 2229
AY150312.1 CCTCCCGTGACTCAAGAGCCTTTGGGCAAAGACTCGGTCCCTCTGACCGCCTTCTCGCTG
2340 ** ****.* ***.********
Trang 21ON584463.1 -CCG 2232
AY150312.1 GTCGATTTAAAAGCTTGGGTCAAAAGCTACCCACGGTGGACACCACCACCCCCTCCACCA
2640 **
ON584463.1
GAATTCCAGTCTCGGAAGGCGGCGTCTGTTAGGAGTCTTCCAGAGGGCAGGCCTTTTCCT 2292AY150312.1
AGGGTTCAACCTCGAAAAACAAAGTCTGTCAAGAGCTTGCCAGAGGACAAGCCTGTCCCC 2700 * ** ****.** * ****** *.*** * *******.**.**** * **
ON584463.1
GCTCCGCGCAGGAAGATCAGGTCCAGGCGTAGTAGTTTGGATTTACCGGGCGACAACTTC 2352AY150312.1
GCTCCGCGCAGGAAGGTCAGATCCGGTTGCGGCAGCCCGGTTTTAATGGGCGACAATGTC 2760 ***************.****.***.* * * ** **:**** ********* **
ON584463.1 CCTGACAGCTGGGAAGGCTTGGCTG - 2377
AY150312.1 CCTAACGGTTCGGAAGATTTGACTGTCGGTGGTCCCCTCAATTTTCCGACACCATCCGAG
2820 ***.**.* * ***** ***.***
ON584463.1 -GTGATCCCTTCCATCCTCCAGCCCTG -CCTAAACCA 2412AY150312.1
CCGATGACACCTATGAGTGAGCCCGTACTTACGCCCGCGCTGCAACGTGTCCCCAAGTTG 2880 **** *** *.*:*.* **.** *** ** **
ON584463.1
GTGGCACGTTCGAGTGGATCTGTGCCTGTCCCTGCACCGCGCAGAACTGTGTCCCGACTG 2472AY150312.1
ATGACACCTTTGGATGGGTCGGCCCCGGTTCCTGCACCGCGTAGAACTGTGTCCCGACCG 2940 **.*** ** * ***.** * ** ** *********** **************** *
ON584463.1
AAGCCGTCACCGGTAGTGTCAACCCCTGTGCCCGCACCACGATGTGGGTTTCGGCAGGTG 2532AY150312.1 ATGACGCCCTTGAGTGAGCCAATTTTTTTGTCTGCACCGCGACACAAATTTCAGCAGGTG
3000 *:*.** * * :*:* *** * ** * *****.*** ****.*******
ON584463.1
GAAGGAATGAACCTGACGGTGGGGACTCCAGCGTGCCAGGATGAGCTCTTCGATTTATCC 2592AY150312.1
GAAGAAGCGAATCCGGCGACAACAACGCTGACGCACCAGAACGAGCCTCTGGATTTGTCT 3060 ****.* *** * *.** ** * ** ****.* **** * *****.**
ON584463.1
GCGTCTTTGCAGACTGAATATGAGGCTTCCCCTTTGGTACTACCACAGAGTGAGGACACC 2652AY150312.1 GCGTCTTCACAAACGGAATATGAGGCTTCCCCCCTAGCATCATCGCAGAACATGAGCATC
3120 ******* **.** ***************** *.* * * *.**** .:* ** *
ON584463.1
CTGGCAGTAAGGAGACGAGAAGCTGAGGAAGTTCTGAGCGAAACTTCTGACTTGCCGGAC 2712AY150312.1
CTGGAGGCGGGGGGGCAAGAAGCTGAGGAAGTTCTGAGTGAAATCTCGGATATACTAAAT 3180
Trang 22**** * **.*.*.********************* **** ** ** :*.* *
ON584463.1
GGCATCAAGCTGACGCCCGTGTCTCCAAGCAGTTCCCTGTCAAACATAGAGATCACACGC 2772AY150312.1 GACACCAGCCCTGCACCTGTGTCATCAAGCAGCTCCCTGTCAAGTGTTAAGATTACACGC
3240 *.** ** * *.** *****: ******* ********** .*:.**** ******
ON584463.1
CCAAAGTATTCAGCTCAAGCTATCATTGACACAGGCGGGCCCTGTTGTGGGCACCTCCGA 2832AY150312.1
CCAAAATACTCAGCTCAAGCCATCATCGACTCTGGCGGGCCTTGCAGTGGGCACCTCCAA 3300 *****.** *********** ***** ***:*:******** ** :************.*
ON584463.1
GAGGTGAAGGAAAAGTACCTCAATGTGATGCGTGAGGCATGCGATGCTACCAAGCTTGAT 2892AY150312.1
AAGGAAAAAGAAGCATGCCTCAGCATCATGCGTGAGGCTTGTGATGCGTCCAAGCTTAGT 3360 ***:.**.*** *.***** .* ***********:** ***** :******** *
ON584463.1
GACCCTGCCACACAAGAATGGCTTTCCCGTATGTGGGACAGGGTAGACATGCTAACCTGG 2952AY150312.1
GATCCTGCTACGCAGGAATGGCTTTCTCGCATGTGGGATAGGGTTGACATGCTGACTTGG 3420 ** ***** **.**.*********** ** ******** *****:********.** ***
ON584463.1 CGCAACACGTCCATCTTTCAGGCGCCTTTCACTTTGGCTGACAAGTTCAAGCTCCTCCCG
3012AY150312.1 CGCAACACGTCTGCTTACCAGGCGTTTCGCACCTTAAATGGCAGGTTTGAGTTTCTCCCA
3480 *********** *: ****** * *** ** **.**.*** ** * *****
ON584463.1 AAGATGATACTCGAAACACCTCCGCCTTACCCTTGTGGGTTTGTAATGATGCCCCGCACA
3072AY150312.1
AAGATGATTCTCGAGACACCGCCGCCCCACCCGTGCGGGTTTGTGATGTTACCTCACACG 3540 ********:*****.***** ***** **** ** ********.***:*.** *.***
ON584463.1
CCAACACCTTCTATAGGTGCGGAAAGCGACATCACCATTGGTTCAGTTGCCACTGAGGAT 3132AY150312.1
CCTGCACCTTCCGTGAGCGCAGAGAGTGATCTCACCATTGGTTCAGTAGCCACTGAGGAT 3600 **:.******* * * **.**.** ** ****************:************
ON584463.1
GTTCCGCGCATTCTCGGGGAGGCGCAAGGTGTTTACAAAACAATCGCACATGAACCCTCA 3192AY150312.1
GTTCCACGCATCCTCGGGAAAATAGGAGACACCGGCGAGCTGCTTAACCAGGGTCCCTCG 3660 *****.***** ******.* ** *.* * ** *.:*****
ON584463.1
GCACCTTTCGCAGGTGGACCGACAGATGGCCGACCTGCTGGAGAACCCCAAACACAAACT 3252AY150312.1
GCACCCTTCAAGGGAGGACCGGTCTGTGACCAACCTGCCAAAAATTCCCGGATGTCGCCG 3720 ***** *** **:****** .**.**.****** *.*: *** * *
Trang 23ON584463.1
CCTCCCGCAGGTGCAAGTGGCGTCGGCTTGGTTTCGGATTCTGAAGAATCGCCAGAGC 3310AY150312.1 CGGGAGTCTGACGAGAGCATAATAGCTCCGCCCGCAGATACAGGTGG
CGCCGGCTCAT 3778
* *:* * ** *.* * *.***:*:*.:* ****.* *
ON584463.1
TCGCTGGCTCGCCGCCTCCAAACGGTGCCGACGCTGGCGGAGGGGGACCGTTGCATGCGG 3370AY150312.1
TCACTGATTTGCCGTCTTCGGATAGTGTGGATGCGAACGGAGGGGGGCCGTTAAGAACGG 3838 **.*** * **** ** * * *** ** ** *********.***** :.***
ON584463.1
TCACGAAGAGGGCTGAGAGGTTCTTTGATCAGCTAAGCAGACGGGTTTTCGGCTTCGCTT 3430AY150312.1 TAAAAACAAAAGCAGGAAGGCTCTTGGACCAACTGAGCTGTCAGGTTTTTAGCCTCGTTT
3898 *.* * * **:* *** **** ** **.**.***:*:*.****** ** *** **
ON584463.1 CCCATCTCCCTATCTTCTTCTCGCGCCTTTTTAGGTCTGACGGTCATTATGCTCCGGGTG
3490AY150312.1 CCCATCTCCCTGTTTTCTTCTCACACCTCTTCAAATCTGACAGTGGTTATTCTCCGGGTG
3958 ***********.* ********.*.*** ** * ******.** **** *********
ON584463.1 ATTGGGGGTTTGCAGCTTTCACTTTATTGTGCCTCTTTCTATGTTACAGTTACCCAGCTT
3550AY150312.1 ATTGGGGTTTTGCAGCTTTTACTCTATTTTGCCTCTTTCTGTGTTACAGCTACCCATTCT
4018 ******* *********** *** **** ***********.******** ****** *
ON584463.1
TCGGCATCGCTCCCTTATTGGGTGTATTTTCTGGGTCTCCTCGGCGCGTCCGCATGGGGG 3610AY150312.1 TCGGCTTTGCTCCCCTCTTGGGTGTATTTTCTGGGTCTTCTCGGCGTGTGCGAATGGGGG
4078 *****:* ****** *.********************* ******* ** **.*******
ON584463.1 TCTTTGGTTGTTGGTTGGCTTTCGCTGTTGGTTTGTTCAAACCTGTGCCCAACCCAGTCG
3670AY150312.1 TTTTTGGCTGCTGGTTGGCTTTCGCTGTTGGTCTGTTCAAGCCTGTGTCCGACCCAGTCG
4138
* ***** ** ********************* *******.****** **.*********
ON584463.1 GTACTGCTTGTGAATTTGACTCGCCGGAGTGTAGAGATGTCCTTCATACTTTTGAGCTTC
3730AY150312.1 GCACTGCTTGTGAGTTTGACTCGCCAGAGTGTAGGAACGTCCTTCATTCTTTTGAGCTTC
4198
* ***********.***********.******** * *********:************
ON584463.1
AGCAACCTTGGGACCCTGTTCGCAGCCTTGTGGTGGGCCCCATCGGTCTCGGTCTTGCCA 3790AY150312.1
TCAAACCTTGGGACCCTGTCCGCAGCCTTGTTGTGGGCCCCGTCGGTCTCGGCCTTGCCA 4258 : **************** *********** *********.********** *******
Trang 24ON584463.1
TTCTTGGCAGGTTACTGGGCGGGGCACGCTACGTCTGGTTGCTTTTGCTTAGGCTTGGCA 3850AY150312.1 TTCTTGGCAGGTTACTGGGCGGGGCACGCTACGTCTGGCACTTTTTGCTTAGGTTTGGTA
4318 ************************************** : *********** **** *
ON584463.1
TCGTTTCAGACTGTATCCTGGCTGGAGCTTATGTGCTTTCGCAAGGCAGGTGTAAAAGGT 3910AY150312.1 TTGTTGCAGATTGTATCTTGGCTGGAGCTTATGTGCTTTCTCAAGGTAGGTGTAAAAAGT
4378
* *** **** ****** ********************** ***** **********.**
ON584463.1
GCTGGGGATCTTGTGTAAGAACCGCCCCAAGCGAGGTTGCCTTCAACGTGTTTCCCTTCA 3970AY150312.1 GCTGGGGATCTTGTGTAAGAACTGCTCCTAATGAGATCGCCTTCAACGTGTTCCCTTTCA
4438 ********************** ** **:* ***.* ************** ** ****
ON584463.1
CACGTGCGACCAGGTCATCGCTCATCGATTTGTGCAATCGATTCTGTGCACCAAAGGGCA 4030AY150312.1
CACGTGCGACCAGGTCGTCACTCATCGACCTGTGCGATCGGTTTTGTGCGCCAAAAGGCA 4498 ****************.**.******** *****.****.** *****.*****.****
ON584463.1
TGGATCCTATATTCCTCGCCACTGGATGGCGCGGATGCTGGATCGGCCAAAGCCCCGTTG 4090AY150312.1
TGGACCCCATTTTTCTCGCCACTGTGTGGCGTGGGTGCTGGACCGGCCGGAGTCCCATTG 4558 **** ** **:** ********** ***** **.******* ***** ** ***.***
ON584463.1
AGCAACCATCTGAGAAACCCATCGCGTATGCCCAATTGGATGAAAAGAAGATCACGGCCA 4150AY150312.1
AGCAACCCTCTGAAAAACCCATCGCGTTCGCCCAGCTGGATGAAAAGAGGATTACGGCTA 4618 *******.*****.*************: ***** ************.*** ***** *
ON584463.1
GGACTGTGGTTGCCCAACCTTACGACCCCAATCAAGCTGTGAAGTGCTTACGAGTTTTGC 4210AY150312.1 GAACTGTGGTCGCTCAGCCTTATGATCCTAACCAAGCCGTAAAGTGCTTGCGGGTATTAC
4678 *.******** ** **.***** ** ** ** ***** **.********.**.**:**.*
ON584463.1
AGGCGGGTGGGGCGATGGTGGCTGAGGCGGTCCCAAGAGTGGTTAAAGTCTCTGCTGTCC 4270AY150312.1
AGGCGGGTGGGGCGATGGTGGCCGAGGCAGTCCCAAAAGTGGTCAAGGTTTCCGCTATTC 4738 ********************** *****.*******.****** **.** ** ***.* *
ON584463.1
CATTTCGAGCCCCCTTCTTCCCCACCAACGTCAAAGTCGATCCTGAATGCAGGGTCGTGG 4330AY150312.1 CATTCCGAGCTCCTTTTTTTCCCGCTGGAGTGAAAGTTGATCCTGAGTGCAGAATCGTGG
4798 **** ***** ** ** ** ***.* ** ***** ********.***** ******
Trang 25ON584463.1 TTGACCCAGACACCTTTACAACCGCTCTCCGGTCCGGCTACTCCACCACAAACCTTATTC
4390AY150312.1 TTGACCCCGATACTTTTACTACAGCCCTCCGGTCTGGCTATTCCACCACGAACCTCGTCC
4858 *******.** ** *****:**.** ******** ***** ********.***** * *
ON584463.1
TTGGCGTGGGAGACTTCGCTCAGTTGAATGGGTTGAAAATCAGACAAATCTCTAAGCCAT 4450AY150312.1 TTGGTATGGGGGACTTTGCCCAGCTGAATGGACTAAAGATCAGGCAAATTTCCAAGCCTT
4918 **** ****.***** ** *** ******* *.**.*****.***** ** *****:*
ON584463.1
CAGGGGGGGGGCCGTACCTCGTGGCGGCCTTGCACGTCGCTTGCTCGATGGCCTTGCATA 4510AY150312.1
CAGGAGGAGGCTCACACCTCGTTGCTGCCTTGCATGTTGCCTGCTCGATGGCGTTGCACA 4978 ****.**.** * ******* ** ******** ** ** *********** ***** *
ON584463.1
TGCTCGCTGGGATTTATGTCACTGCGGTGGGTTCTTGTGGCTCTGGCACTAACGACCCAT 4570AY150312.1 TGCTTGCTGGTGTTTATGTAACTGCAGTGGGGTCTTGCGGTACCGGTACCAACGATCCGT
5038 **** ***** *******.*****.***** ***** ** :* ** ** ***** **.*
ON584463.1
GGTGCACCAACCCGTTTGCCGTCCCTGTCTATGGGCCTGGCTCTCTTTGCACGTCCAGGT 4630AY150312.1 GGTGCACTAACCCGTTTGCCGCCCCTGGCTACGGACCTGGCTCTCTTTGCACGTCCAGAT
5098 ******* ************* ***** *** **.***********************.*
ON584463.1
TGTGCATTTCCCAGCAAGGCCTTACACTACCTTTGACGGCGCTTGTGGCGGGGTTTGGCA 4690AY150312.1 TGTGCATCTCCCAACACGGTCTCACCTTGCCCTTGACAGCACTTGTGGCGGGATTCGGCC
5158 ******* *****.**.** ** ** *.** *****.**.***********.** ***
ON584463.1 TCCAGGAAGTTGCTTTGGTCGTTTTAATCTTTACTTCTGTATGTGGCATGGCCCACAGGT
4750AY150312.1 TTCAAGAGATTGCCTTGGTCGTTTTGATTTTTGTCTCCATGGGGGGCATGGCTCATAGGT
5218
* **.** **** ***********.** *** ** * * ******** ** ****
ON584463.1 TGAGTTGCAAGGCTGATGTAATGTGCATCCTACTTGCTATTGTTAGCTATGTCTGGGCGC
4810AY150312.1 TGAGTTGTAAGGCTGACATGCTGTGCATCTTACTCGCAATAGCCAGCTATGTTTGGGTAC
5278 ******* ******** * ******** **** **:**:* ******** **** *
ON584463.1 CTCTCGCTTGGTTGCTTTGTACGTTCCCTTGCTGGTTGCGCTGGTTTTCTTTGCATCCCC
4870AY150312.1 CTCTTACCTGGTTGCTTTGTGTGTTTCCTTGTTGGTTGCGCTGGTTCTCTTTGCACCCCC
5338 **** * ************ *** ***** ************** ******** ****
Trang 26ON584463.1 TTACCATTCTATGGTTGGTGTTTTTCTTGATTTCCGTGAACATGCCCTCAGGAATCTTGG
4930AY150312.1 TCACCATCCTATGGTTGGTGTTTTTCTTGATTTCTGTAAATATACCCTCGGGAATCTTGG
5398
* ***** ************************** **.** **.*****.**********
ON584463.1 CTTTAGTGTTGTTGATTTCTCTCTGGCTTCTTGGTCGCTACACCAATGTTGCTGGCCTAG
4990AY150312.1 CCGTGGTGTTATTGGTTTCTCTCTGGCTTTTAGGTCGTTATACTAACATTGCTGGTCTCG
5458
* *.*****.***.************** *:***** ** ** ** ******* **.*
ON584463.1
TCACCCCTTACGACATTCACCACTACACCAATGGCCCCCGCGGCGTCGCCGCCTTAGCCA 5050AY150312.1 TCACCCCCTATGATATTCATCATTACACCAGTGGCCCCCGCGGTGTCGCCGCCTTGGCTA
5518 ******* ** ** ***** ** *******.************ ***********.** *
ON584463.1
CAGCTCCTGATGGGACCTATTTGGCAGCTGTCCGCCGCGCTGCGTTGACTGGCCGTACCA 5110AY150312.1
CCGCACCAGACGGAACCTACTTGGCTGCCGTCCGCCGCGCTGCGTTGACTGGTCGTACCA 5578 *.**:**:** **.***** *****:** *********************** *******
ON584463.1
TGCTGTTCACGCCGTCTCAACTTGGATCGCTCCTTGAGGGCGCCCTTAGAACCCAAAAGC 5170AY150312.1 TGCTGTTTACCCCGTCTCAGCTCGGGTCCCTCCTTGAGGGCGCTTTCAGAACTCAAAAGC
5638 ******* ** ********.** **.** ************** * ***** *******
ON584463.1
CTTCATTGAATACCGTCAATGTGGTTGGGTCTTCCATGGGCTCCGGCGGGGTGTTCACCA 5230AY150312.1
CCTCACTGAACACCGTCAATGTGGTCGGGTCCTCCATGGGCTCTGGCGGAGTGTTCACTA 5698
* *** **** ************** ***** *********** *****.******** *
ON584463.1
TTGACGGGAAAATCAAATGTGTAACCGCCGCACACATCCTTACGGGCAACTCTGCTAGGG 5290AY150312.1
TTGACGGGAAAATCAAGTGCGTAACTGCCGCACATGTCCTTACGGGTAACTCAGCTAGGG 5758 ****************.** ***** ******** ********** *****:*******
ON584463.1 TATCTGGGGTTGGTTTCAACCAAATGTTGGATTTCGACGTGAAAGGGGATTTTGCCATAG
5350AY150312.1 TTTCCGGGGTCGGCTTCAATCAAATGCTTGACTTTGATGTAAAAGGGGACTTCGCCATAG
5818 *:** ***** ** ***** ****** * ** ** ** **.******** ** *******
ON584463.1
CCGATTGTCCGGATTGGCAAGGAGCCGCTCCCAAGTCCCAGTTCTGTGAGGATGGGTGGA 5410AY150312.1
CTGATTGCCCGAATTGGCAAGGGGCTGCTCCCAAGGCCCAGTTCTGCGAGGATGGGTGGA 5878
* ***** ***.**********.** ********* ********** *************
Trang 27ON584463.1
CTGGCCGCGCTTATTGGCTGACATCCTCCGGCGTCGAACCTGGCGTCATTGGGAGGGGAT 5470AY150312.1
CTGGTCGCGCCTATTGGCTGACATCCTCTGGCGTCGAACCCGGTGTCATTGGGAATGGGT 5938 **** ***** ***************** *********** ** ********** **.*
ON584463.1
TTGCTTTTTGTTTCACCGCGTGTGGCGATTCCGGGTCCCCAGTGATCACCGAGGCCGGAG 5530AY150312.1 TTGCCTTCTGCTTCACCGCGTGTGGCGATTCTGGATCCCCAGTGATTACCGAAGCCGGTG
5998 **** ** ** ******************** **.*********** *****.*****:*
ON584463.1
AACTTGTTGGTGTCCACACGGGATCAAACAAACAAGGAGGAGGTATTGTCACGCGCCCTT 5590AY150312.1
AGCTTGTCGGCGTTCACACAGGATCAAACAAACAAGGAGGAGGCATTGTCACGCGCCCCT 6058 *.***** ** ** *****.*********************** ************** *
ON584463.1 CAGGCCGGTTTTGTAATGTGACACCTACCAAATTAAGTGAATTGAGTGAATTCTTCGCTG
5650AY150312.1 CAGGCCAGTTTTGTAATGTGACGCCCATCAAGCTGAGCGAGTTGAGTGAATTCTTCGCTG
6118 ******.***************.** * *** *.** **.*******************
ON584463.1
GACCCAAGGTCCCGCTCGGCGACGTGAAGGTTGGCAACCACATAATTAGAGATGTGTGCG 5710AY150312.1
GACCTAAGGTACCGCTCGGTGATGTGAAGATTGGCAGTCACATAATTAAAGACACATGCG 6178 **** *****.******** ** ******.****** **********.*** ****
ON584463.1
AGGTGCCTTCTGATCTTTGTGCCTTGCTTGCTGCCAAACCTGAGCTGGAGGGGGGCCTCT 5770AY150312.1 AGGTGCCTTCAGATCTTTGTGCCCTGCTTGCTGCCAAACCTGAACTGGAAGGAGGCCTTT
6238 **********:************ *******************.*****.**.***** *
ON584463.1
CCACCGTCCAACTTCTGTGTGTGTTCTTTCTCCTGTGGAGAATGATGGGGCATGCCTGGA 5830AY150312.1 CCACCGTTCAACTTCTGTGTGTGTTTTTCCTCCTGTGGAGAATGATGGGGCATGCTTGGA
6298 ******* ***************** ** ************************** ****
ON584463.1 CGCCTTTGGTTGCTGTGGTTTTTTTTATATTGAATGAGATACTCCCAGCTGTCCTGGTCC
5890AY150312.1 CGCCCTTGGTTGCTGTGGGGTTTTTTATCCTGAATGAAATTCTCCCAGCTGTCCTGGTCC
6358 **** ************* ******** *******.**:*******************
ON584463.1 GGAGTGTCTTCTCCTTTGGCATGTTTGCGTTGTCTTGGTTTACACCATGGTCTGCGCAAG
5950AY150312.1 GGAGTGTTTTCTCCTTTGGGATGTTTGTGCTATCTTGGCTCACACCATGGTCTGCGCAAG
6418 ******* *********** ******* * *.****** * *******************
Trang 28ON584463.1 TTCTGATGATCAGGCTTCTAACAGCTGCTCTTAACAGGAACAGGGGGTCACTCTTTTTCT
6010AY150312.1 TCCTGATGATCAGGCTTCTGACAGCAGCCCTTAACAGGAACAGATTGTCTCTTGGTTTTT
6478
* *****************.*****:** ************** ***:** *** *
ON584463.1
ACATCCTCGGTGCAGTGACCGGATTTGTCGCAGATCTTGCGGTGACTCAGGGGCATCCGC 6070AY150312.1 ACAGTCTTGGTGCAGTAACCAGTTTTGTCGCAGATCTTGCGGTAACTCAAGGGCATCCGT
6538 *** ** ********.***.*:********************.*****.*********
ON584463.1 TGCAGGCAGCGATGAACTTGAGCACCTATGCTTTCCTGCCTCGAATGACGGTTGTCACTT
6130AY150312.1 TACAGGTAGTAATGAACTTAAGCACCTATGCCTTCCTGCCTCGGATGATGGTTGTGACCT
6598 *.**** ** ********.*********** ***********.**** ****** ** *
ON584463.1 CACCGGTCCCGTTGCTTGCTTGTGGTGTTGTGCACCTTCTTGCCATAATTTTGTACCTGT
6190AY150312.1 CGCCAGTCCCAGTGATCGCGTGTGGTGTTGTGCACCTCCTTGCCATAATTTTGTACTTGT
6658 *.**.***** **.* ** ***************** ****************** ***
ON584463.1 TCAAGCACCGTAGCCTGCATTATGTTCTTGTTGGCGATGGAGTGTTCTCTAGCGCCTTCT
6250AY150312.1 TTAAGTACCGCTGCCTTCACTATGTCCTTGTTGGCGATGGGGTGTTCTCTTCGGCTTTCT
6718
* *** **** :**** ** ***** **************.*********: ** ****
ON584463.1
TCTTGCGATATTTTGCTGAGGGAAAGTTGAGGGAAGGGGTGTCGCAGTCCTGCGGAATAA 6310AY150312.1
TCTTGCGATACTTTGCTGAGGGAAAGTTGAGGGAAGGGGTGTCGCAATCCTGCGGGATGA 6778 ********** ***********************************.********.**.*
ON584463.1
ATCACGAGTCACTGACTGGCGCTCTCGCTATGAGACTCGATGACGAAGACCTGGATTTCC 6370AY150312.1
GTCATGAGTCGCTGACTGGTGCCCTCGCCATGAGACTCACTGACGAGGACTTGGATTTCC 6838 *** *****.******** ** ***** ********* ******.*** *********
ON584463.1 TTGCAAAACTAACTGATTTTAAGTGCTTTGTTTCCGCATCCAACATGAGGAATGCGGCGG
6430AY150312.1 TTACGAAATGGACTGATTTTAAGTGCTTTGTTTCTGCGTCCAACATGAGGAATGCAGCGG
6898 **.*.*** *********************** **.*****************.****
ON584463.1
GCCAGTTTATCGAGGCTGCTTATGCCAAAGCACTTAGGGTGGAACTTGCCCAGCTGGTGC 6490AY150312.1 GCCAATTTATCGAGGCTGCTTATGCAAAAGCACTAAGAATTGAACTTGCTCAGTTGGTAC
6958 ****.********************.********:** * ******** *** ****.*
Trang 29ON584463.1
AAGTTGACAAGGTTCGGGGCGTTTTGGCTAAGCTTGAAGCTTTTGCTGATACCGCAACGC 6550AY150312.1
AGGTTGACAAGGTCCGAGGCACCTTGGCCAAACTTGAGGCTTTTGCCGATACCGTGGCAC 7018 *.*********** **.*** ***** **.*****.******** ******* *.*
ON584463.1
CCCAACTCTCACCTGGCGACATTGTCGTTGCTCTTGGCCACACGCCCGTCGGCAGCATCT 6610AY150312.1 CCCAACTCTCGCCCGGTGACATTGTTGTTGCCCTTGGCCACACGCCTGTTGGCAGCATCT
7078 **********.** ** ******** ***** ************** ** **********
ON584463.1
TTGACTTAAAAGTAGGCAGCACCAAGCACACCCTACAAGCCATCGAGACCAGAGTCCTCG 6670AY150312.1 TCGACCTAAAGGTTGGTAGCACCAAGCACACTCTCCAAGCCATTGAAACTAGAGTCCTTG
7138
* *** ****.**:** ************** **.******** **.** ******** *
ON584463.1
CTGGGTCCAGAATGACCGTGGCGCGCGTCGTTGATCCGACTCCCACACCTCCACCCGTGC 6730AY150312.1
CCGGGTCCAAAATGACTGTAGCGCGTGTCGTTGACCCAACCCCTGCACCCCCACCCGTAC 7198
* *******.****** **.***** ******** **.** ** **** ********.*
ON584463.1
CCGTGCCCATTCCTCTCCCGCCGAAGGTTTTAGAGAACGGCCCCCGTGCCTGGGAGGATG 6790AY150312.1
CTGTGCCCATCCCTCTCCCACCGAAAGTTCTGGAGAACGGTCCCAACGCCTGGGGGGATG 7258
* ******** ********.*****.*** *.******** *** *******.*****
ON584463.1
AGGACCGCCTAAACAAGAAAAAACGGCGCAAAATGGAGGCCGTTGGCATTTATGTCATGG 6850AY150312.1
AGGACCGTTTGAACAAGAAGAAGAGGCGCAGGATGGAAGCCGTCGGCATCTTTGTCATGG 7318 ******* *.********.** ****** *****.***** ***** *:********
ON584463.1 ATGGGAAAAAGTACCAAAAATTTTGGGACAAAAATTCTGGTGATGTGTTCTATGAAGAAG
6910AY150312.1
ACGGGAAAAAGTACCAGAAATTTTGGGACAAGAATTCCGGTGATGTGTTTTATGAGGAGG 7378
* **************.**************.***** *********** *****.**.*
ON584463.1
TCCACGACAACACAGACGCGTGGGAATGCCTTAGAACCGACGACCCTGCCGACTTGGATC 6970AY150312.1
TCCACAATAGCACAGACGAGTGGGAGTGCCTTAGAGCTGGCGACCCTGCCGACTTTGACC 7438 *****.* *.********.******.*********.* *.*************** ** *
ON584463.1
CTGAGAAGGGGACTTTGTGCGGGCGCCTCATCATAGAGAATAAACCTTACCAAGTTTATG 7030AY150312.1 CTGAGACAGGGGTTCAGTGTGGGCATATCACCATTGAAGATAGGGTTTACAATGTCTTCA
7498 ****** *** * :*** **** .*** ***:** *** ****.*:** *: