polymerase-endonuclease amplification reaction (pear) and its potential applications

Polymerase-Endonuclease Amplification Reaction (PEAR) and its potential

applications

Xiaolong WangCollege of Life Sciences, Ocean University of

China

[email protected]

Nucleic Acids Enzymes and Enzymes in Human Disease (NAEEHD)Jun 20, 2011, NanKai University

mailto:[email protected]

Outline

I. DNA amplification technologies

II. What is PEAR?

III. Potential applications of PEAR

1. Large-scale preparation of oligonucleotides

2. Preparation of repeat DNA sequences.

3. Detection and quantification oligonucleotides

and small RNAs

I. DNA amplification technologies:1. Thermocycling reactions1) PCR: Polymerase Chain Reaction, Science,1985

2) LCR: Ligase Chain Reaction, PNAS,1991

3) PEAR: Polymerase-Endonuclease Amplification Reaction, PLoS One, 2010

2. Isothermal reactions4) SDA: Strand Displacement Amplification, PNAS,1992

5) RCA: Rolling circle amplification, Nature Genetics,1998

6) LAMP: loop-mediated isothermal amplification, NAR, 2000

7) HDA: helicase-dependent amplification, EMBO reports, 2004

8) EXPAR: Exponential amplification reaction, PNAS, 2003

Outline


II. What is PEAR?





and small RNAs

X’ R’ X’

X’ R’ X’

X’ R’ X’

X

X R X

X’ R’ X’

X

X

X’ X’

X X

Bst polymerase

Cleaving

Probe Target

Strand

displacement

Nikase

Annealing

XAnnealing

EXPAR: Exponential amplification reaction

Van Ness J, Van Ness LK, Galas DJ. 2003. Isothermal reactions for the amplification of oligonucleotides, PNAS, 100 (8): 4504–4509

X’ R’ X’

X’ R’ X’

X R X R X

X’ R’ X’

X

X R X

X’ R’ X’

X

X

X

X’ X’

X X

X’ R’ X’

X R X

X’

X’

X’ R’ X’

X R X

X’ R’ X’ R’ X’

X’ R’ X’

X R X

Elongation Taq

polymerase

Cleaving

Probe Target

Den

atu

rati

on

PspGI

Annealing

X

Annealing

Denaturation

Denaturation

Annealing(slipping)

Elongation

Cle

avin

g+

Annealing

Annealing

Wang, Xu and Gou, PLoS One, 2010, 5 (1):e8430

PEAR: Polymerase-Endonuclease Amplification Reaction

Thermostable endonucleasePspGI (NEB R0611S 1,000U 10U/uL $58.00)

Dr. Shuang-yong Xu Senior ScientistNew England Biolabs

Morgan R, Xiao J, Xu S (1998) Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction-modification system in Escherichia coli. Appl Environ Microbiol 64(10): 3669–3673.

http://www.neb.com/nebecomm/researchScientist.asp?id=SXu


Target:PspGI:

Polymerase:

+++++----HLH--HH--++-+-+-+-

M123456789bp

100

40

20

10

M1234567

Input target concentration (nM) 1 10-1 10-2 10-3 10-4 10-5 10-6

bp

10020

10

Outline


II. What is PEAR?





and small RNAs

Many diseases are caused by genes

Gene therapy a possible solution

Repairing a damaged good gene

Inhibiting a bad or overexpressed gene

I. Gene knockoutII. Gene KnockdownmRNA

1) RNAi2) miRNA3) Antisense oligonucleotides

miRNA4) Antisense oligonucleotides5) miRNA sponge

RNAi is effective for mRNA knockdown

miRNA is a endogenous small RNA

How miRNA works?

Antisense miRNA oligonucleotide (AMO)

J Weiler et al Anti-miRNA oligonucleotidesGene Therapy (2006) 13, 496–502

* Antisense oligonucleotide (ASO)

Fomivirsen (Vitravene) — the first and only antisense antiviral drug approved by FDA

Fomivirsen (ISIS 2922)

$63.87 USD

Modified ASO

Chemically modified oligonucleotide analogs that have been used as anti-miRNA inhibitors (AMOs).

Locked Nucleic Acid (LNA)

http://www.pnas.org/content/97/10/5633/F1.large.jpg

Jesper Wengel Professor, University of Southern Denmark

Jesper Wengel is professor of bioorganic chemistry at the University of Southern Denmark and Director of the Nucleic Acid Center, a research center of excellence focused on nucleic acid chemical biology.

He is the co-inventor of LNA (locked nucleic acid) and inventor of UsiRNA, and he is co-founder of RiboTask, a biotech company focused on developing and marketing novel technologies for gene silencing.

Locked Nucleic Acid (LNA)

Wahlestedt C et al. PNAS 2000;97:5633-5638

SCIENCE VOL 327 8 JANUARY 2010

Why amplify oligonucleotides?

Almost all oligonucleotides are chemical

synthesized, but large-scale synthesis of

oligonucleotides is still difficult,

1. Purity: n-1 deletions and failure sequences;

2. Large-scale HPLC purification is difficult;

3. Uses hazardous Chemicals;

4. Expensive instruments.

Chemical Synthetic modified Antisense Oligonucleotides is extremely expensive!

A possible way out:

PEAR for large-scale Enzymatic Production of Antisense Oligonucleotides

PEAR for Enzymatic Production of Oligos

Is it feasible to synthesize modified ASO by PEAR?

Outline


II. What is PEAR?





and small RNAs

PEAR is a minimal DNA replication system, to study the origin and evolution of repetitive DNA in genome, as well as the origin and evolution of genetic material and life.

The repeat PEAR product DNA can be transferred into cells or organisms to study

1) Function of repeat DNA sequences.

2) Synthetic biology

3) Molecular evolution

Potential applications of Repeat DNA

X’ R’ X’

X’ R’ X’

X R X R X

X’ R’ X’

X

X R X

X’ R’ X’

X

X

X

X’ X’

X X

X’ R’ X’

X R X

X’

X’

X’ R’ X’

X R X

X’ R’ X’ R’ X’

X’ R’ X’

X R X

Elongation

dNTPsTaq polymerase

Cleaving

Probe Target

De

na

tura

tio

n

PspGI

DenaturationAnnealing

X

Annealing

Denaturation

Denaturation

Annealing(slipping)

Elongation

Cle

av

ing

+

Annealing

Annealing

Outline


II. What is PEAR?





and small RNAs

I. Real-time detection methods1. Thermocycling reactions1) PCR: Polymerase Chain Reaction2) LCR: Ligase Chain Reaction3) PEAR: Polymerase-Endonuclease Amplification Reaction

2. Isothermal reactions4) SDA: Strand Displacement Amplification5) RCA: Rolling circle amplification6) LAMP: loop-mediated isothermal amplification7) HDA: helicase-dependent amplification 8) EXPAR: Exponential amplification reaction

M’

m

mTarget miRNA

Poly A TailingPAP

Reverse transcriptionOligo dTdNTPsReverse transcriptase

Real-time PCR

(A)nAAAAA

(A)nAAAAATTTTTTTT

m

cDNA

cDNATTTTTTTT

M’

RNase H

Universal Tag

Universal PrimerGene Specific Primer

M’

m

mTarget miRNA

Poly A TailingPAP

Reverse transcriptionOligo dTdNTPsReverse transcriptase

Real-time PEAR

(A)nAAAAA

(A)nAAAAATTTTTTTT

m

cDNA

cDNATTTTTTTT

M’

RNase H

Repeat Probe

M R m

M’ R’ M’

M’ R’ M’

m

M’ R’ M’m

ProbeTarget miRNA

E. Coli Polymerase I

DenaturingAnnealing

Real-time PEAR

Repeat Probe

Labeling PEAR probe and real-time PEAR

X’ R’ X’

X R XX

X’ R’ X’

PspGITaq polymerase

X’ X’

X X

FAM TAMRA FAM TAMRA FAM TAMRA

PspGI

Number of Cycles

Fluorescent intensity

M1234567

Input target concentration (nM) 1 10-1 10-2 10-3 10-4 10-5 10-6

bp

10020

10

Why thermocycling reaction (PCR) outperforms

isothermal reactions?

PCR is much more reliable in most situations:

1) PCR Reaction is tightly controlled by thermocycling, easy and reliable, while isothermal reaction is not controllable once started;

2) Thermocycling helps mixing reaction mixture;

3) Instrument is not a real problem, especially in a

modern molecular biology laboratory;

Conclusion

I. PEAR is a new DNA amplification technology

II. PEAR is Potentially useful for:



3. Detection and quantification

oligonucleotides and small RNAs

Acknowledgements

苟德明博士、教授深圳大学特聘教授Assistant Professor, University of Illinois at Chicago

Dr. Shuang-yong Xu, Senior ScientistRestriction EnzymesNew England Biolabs

期限名称来源2011~2013 聚合酶 -内切酶扩增反应制备反义寡核苷

酸 (81072567)国家自然科学基金

2011~2013 PEAR制备反义寡核苷酸药物(ZR2010HM056)

山东省自然科学基金


Our groupDr. Jianye Zhang, lecturer

Dr. Gang Chen, lecturer

1. PEAR Technology Research Group

(1). Shihua Dong, Jiajun Wu, Benpeng Miao, Zhu Song: Preparation of antisense

oligonucleotides

(2). Ting Xu, Tingyu Zhou, Junfei Ran, Chuanliang Gong, Lei Wei: Preparation of

repetitive sequences：2. DNA+Pro Software Development Group

(1). Yu Fu: Construction of DNA+Pro website

(2) Qi Wang: Programming in Windows/C++ version:

(3) Yue Zhao: Data simulation

(4). Yingbo Niu: analysis human and mammalian genomic protein genes:

(5). Dan Luo: analysis human and mammalian mitochondrial genes:

(6). Rong Jing: analysis virus protein genes and genomes:

(7). Xiaoqing Fu: analysis of bacterial protein genes:

(8). Xiaoyan Liu: analysis of plant protein genes:

Thanks you!

Download this PPT and software in:

www.DNAPlusPro.com.

http://www.dnapluspro.com/

Coding DNA sequencesatg ggg ata aat … tgaatg ata aat agt … tga

Peptide Sequences M G I N … *M I N S … *

Combined DNA-protein Sequences atgM gggG ataI aatN … tga*atgM ataI aatN agtS … tga*

Combined alignmentatgM gggG ataI aatN ---- … tga*atgM gggG ataI ---- agtS … tga*

Translate

Combine

Align

United Codon-aa Sequence Alignment

HV1J3

HV1OY

HV1B1

HV1C4

HV1A2

HV1RH

HV1EL

HV1ND

HV1Z84

HV1MA

HV1ZH

SIVCZ

HV2BE

HV2D1

HV2G1

HV2NZ

HV2CA

HV2D2

5499

54

99

100

8497

100

84

83

55

99

27

17

39

HV1J3

HV1OY

HV1B1

HV1C4

HV1A2

HV1RH

HV1EL

HV1ND

HV1Z84

HV1MA

HV1ZH

SIVCZ

HV2BE

HV2D1

HV2G1

HV2NZ

HV2CA

HV2D2

5499

54

99

100

8497

100

84

83

55

99

27

17

39

HV1J3

HV1OY

HV1B1

HV1C4

HV1A2

HV1RH

HV1EL

HV1ND

HV1Z84

HV1MA

HV1ZH

SIVCZ

HV2BE

HV2D1

HV2G1

HV2NZ

HV2CA

HV2D2

5499

54

99

100

8497

100

84

83

55

99

27

17

39

218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238<HV1J3> I N N S T K D N I K N - - - - D N S T R Y

<HV1B1> I D N - - - - - - - - - - - - - D T T S Y

<HV1C4> I D D N K N T - - - - - - - - T N N T K Y

<HV1A2> I D N A S T T - - - - - - - - T N Y T N Y

<HV1OY> I D - - - - - - - - - - - - - K N D T K F

<HV1RH> I E K G N I S P K N N T S N N T S Y G N Y

<HV1ND> I D N N N - - - - - - - - - R T N S T N Y

<HV1EL> I D N D S - - - - - - - - - S T N S T N Y

<HV1Z84> I D D D N S A N T S - - - - N T N Y T N Y

<HV1MA> I D D S D - - - - - - - - - - - - N S S Y

<HV1ZH> I G G N S S N - - - - - - - - G D S S K Y

<SIVCZ> L G N E N - - - - - - - - - - - - - N T Y

<HV1J3> ataI aatN aatN agtS accT aagK gatD aatN ataI aaaK aatN ---- ---- ---- ---- gatD aatN agtS accT agaR tatY

<HV1B1> ataI gatD aatN ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- gatD actT accT agcS tatY

<HV1C4> ataI gatD gatD aatN aaaK aatN actT ---- ---- ---- ---- ---- ---- ---- ---- accT aacN aacN accT aaaK tatY

<HV1A2> ataI gatD aatN gctA agtS actT actT ---- ---- ---- ---- ---- ---- ---- ---- accT aacN tatY accT aacN tatY

<HV1OY> ataI gatD ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- aagK aatN gatD actT aaaK tttF

<HV1RH> ataI gagE aagK ggtG aatN attI agcS cctP aagK aatN aatN actT agcS aatN aatN actT agcS tatY ggtG aacN tatY

<HV1ND> ataI gacD aatN aatN aatN ---- ---- ---- ---- ---- ---- ---- ---- ---- aggR accT aatN agtS actT aatN tatY

<HV1EL> ataI gacD aatN gatD agtS ---- ---- ---- ---- ---- ---- ---- ---- ---- agtS accT aatN agtS accT aatN tatY

<HV1Z84> ataI gatD gatD gatD aatN agtS gctA aatN accT agtS ---- ---- ---- ---- aatN accT aatN tatY accT aatN tatY

<HV1MA> ataI gatD gatD agtS gatD ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- aatN agtS agtS tatY

<HV1ZH> attI gggGggaG aatN agtS agtS aatN ---- ---- ---- ---- ---- ---- ---- ---- ggtG gatD agtS agtS aaaK tatY

<SIVCZ> ctaL gggG aatN gagE aacN ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- aacN acaT tatY

<HV1J3> ata aat aat agt acc aag gat aat ata aaa aat --- --- --- --- gat aat agt acc aga tat<HV1B1> ata gat aat --- --- --- --- --- --- --- --- --- --- --- --- --- gat act acc agc tat<HV1C4> ata gat gat aat aaa aat act --- --- --- --- --- --- --- --- acc aac aac acc aaa tat<HV1A2> ata gat aat gct agt act act --- --- --- --- --- --- --- --- acc aac tat acc aac tat<HV1OY> ata gat --- --- --- --- --- --- --- --- --- --- --- --- --- aag aat gat act aaa ttt<HV1RH> ata gag aag ggt aat att agc cct aag aat aat act agc aat aat act agc tat ggt aac tat<HV1ND> ata gac aat aat aat --- --- --- --- --- --- --- --- --- agg acc aat agt act aat tat<HV1EL> ata gac aat gat agt --- --- --- --- --- --- --- --- --- agt acc aat agt acc aat tat<HV1Z84> ata gat gat gat aat agt gct aat acc agt --- --- --- --- aat acc aat tat acc aat tat<HV1MA> ata gat gat agt gat --- --- --- --- --- --- --- --- --- --- --- --- aat agt agt tat<HV1ZH> att ggg gga aat agt agt aat --- --- --- --- --- --- --- --- ggt gat agt agt aaa tat<SIVCZ> cta ggg aat gag aac --- --- --- --- --- --- --- --- --- --- --- --- --- aac aca tat

2A

HV1B1

HV1J3

HV1C4

HV1A2

HV1OY

HV1RH

HV1ND

HV1EL

HV1Z84

HV1MA

HV1ZH

SIVCZ

100

73

97

97

100

82

81

95

61

HV1B1

HV1J3

HV1C4

HV1A2

HV1OY

HV1RH

HV1ND

HV1EL

HV1Z84

HV1MA

HV1ZH

SIVCZ

100

73

97

97

100

82

81

95

61

HV1B1

HV1J3

HV1C4

HV1A2

HV1OY

HV1RH

HV1ND

HV1EL

HV1Z84

HV1MA

HV1ZH

SIVCZ

100

73

97

97

100

82

81

95

61

Seq 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

HV1B1 ataI ccaP ataI ---- ---- ---- ---- ---- gatD ---- ---- aatN ---- ---- ---- ---- ---- gatD ---- ---- ---- ---- ---- ---- ---- ---- ---- actT accT agcS tatY ---- ---- ---- acgT ttgL acaT

HV1J3 gtaV ccaP ataI aatN aatN agtS accT aagK gatD ---- ---- aatN ataI ---- ---- aaaK aatN gatD aatN ---- ---- ---- ---- ---- ---- ---- ---- agtS accT agaR tatY ---- ---- ---- agaR ttaL ataI

HV1C4 gaaE ccaP ataI gatD ---- ---- ---- ---- gatD ---- ---- aatN aaaK ---- ---- ---- ---- aatN actT accT ---- ---- ---- ---- ---- ---- aacN aacN accT aaaK tatY ---- ---- ---- aggR ttgL ataI

HV1A2 gtaV ccaP ataI ---- ---- ---- ---- ---- gatD ---- ---- aatN gctA ---- ---- ---- ---- agtS actT ---- ---- ---- ---- ---- ---- ---- ---- actT accT aacN tatY accT aacN tatY aggR ttgL ataI

HV1OY ttaL ccaP ataI ---- ---- ---- ---- ---- gatD aagK ---- aatN ---- ---- ---- ---- ---- gatD ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- actT aaaK tttF ---- ---- ---- aggR ttaL ataI

HV1RH

gtaV ccaP ataI ---- ---- ---- ---- ---- gagEaagK ggtG aatN attI agcS cctP aagK aatN aatN actT agcS ---- ---- ---- ---- ---- ---- aatN aatN actT agcS tatY ggtG aacN tatY acaT ttgL ataI

HV1ND

gtgV ccaP ataI ---- ---- ---- ---- ---- gacD ---- ---- aatN ---- ---- ---- ---- ---- aatN aatN aggR ---- ---- ---- ---- ---- accT aatN agtS actT aatN tatY ---- ---- ---- aggR ttaL ataI

HV1EL gtaV ccaP ataI ---- ---- ---- ---- ---- gacD ---- ---- aatN ---- ---- ---- ---- ---- gatD agtS agtS ---- ---- ---- ---- ---- accT aatN agtS accT aatN tatY ---- ---- ---- aggR ttaL ataI

HV1Z84

gtaV ccaP ataI ---- ---- ---- ---- ---- gatD ---- ---- gatD ---- ---- ---- ---- ---- gatD aatN agtS gctA aatN accT agtS aatN accT aatN tatY accT aatN tatY ---- ---- ---- agaR ttaL ataI

HV1MA

gtaV caaQ ataI ---- ---- ---- ---- ---- gatD ---- ---- gatD ---- ---- ---- ---- agtS gatD aatN agtS ---- ---- ---- ---- ---- ---- ---- ---- ---- agtS tatY ---- ---- ---- aggR ctaL ataI

HV1ZH

gtaV ccaP attI gggGggaG

---- ---- ---- aatN agtS agtS aatN ---- ---- ---- ---- ggtG gatD agtS agtS ---- ---- ---- ---- ---- ---- ---- ---- ---- aaaK tatY ---- ---- ---- agaR ctaL ataI

SIVCZ gtaV aacN ctaL gggG ---- ---- ---- ---- ---- ---- ---- aatN gagE ---- ---- ---- aacN aacN ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- acaT tatY ---- ---- ---- aggR ataI attI

HV1B1 I P I - - - - - D - - N - - - - - D - - - - - - - - - T T S Y - - - T L THV1J3 V P I N N S T K D - - N I - - K N D N - - - - - - - - S T R Y - - - R L IHV1C4 E P I D - - - - D - - N K - - - - N T T - - - - - - N N T K Y - - - R L IHV1A2 V P I - - - - - D - - N A - - - - S T - - - - - - - - T T N Y T N Y R L IHV1OY L P I - - - - - D K - N - - - - - D - - - - - - - - - - T K F - - - R L IHV1RH V P I - - - - - E K G N I S P K N N T S - - - - - - N N T S Y G N Y T L IHV1ND V P I - - - - - D - - N - - - - - N N R - - - - - T N S T N Y - - - R L I

HV1EL V P I - - - - - D - - N - - - - - D S S - - - - - T N S T N Y - - - R L IHV1Z84 V P I - - - - - D - - D - - - - - D N S A N T S N T N Y T N Y - - - R L IHV1MA V Q I - - - - - D - - D - - - - S D N S - - - - - - - - - S Y - - - R L IHV1ZH V P I G G - - - N S S N - - - - G D S S - - - - - - - - - K Y - - - R L I

SIVCZ V N L G - - - - - - - N E - - - N N - - - - - - - - - - - T Y - - - R I IHV1B1 ata cca ata --- --- --- --- --- gat --- --- aat --- --- --- --- --- gat --- --- --- --- --- --- --- --- --- act acc agc tat --- --- --- acg ttg aca

HV1J3 gta cca ata aat aat agt acc aag gat --- --- aat ata --- --- aaa aat gat aat --- --- --- --- --- --- --- --- agt acc aga tat --- --- --- aga tta ata

HV1C4 gaa cca ata gat --- --- --- --- gat --- --- aat aaa --- --- --- --- aat act acc --- --- --- --- --- --- aac aac acc aaa tat --- --- --- agg ttg ata

HV1A2 gta cca ata --- --- --- --- --- gat --- --- aat gct --- --- --- --- agt act --- --- --- --- --- --- --- --- act acc aac tat acc aac tat agg ttg ata

HV1OY tta cca ata --- --- --- --- --- gat aag --- aat --- --- --- --- --- gat --- --- --- --- --- --- --- --- --- --- act aaa ttt --- --- --- agg tta ata

HV1RH gta cca ata --- --- --- --- --- gag aag ggt aat att agc cct aag aat aat act agc --- --- --- --- --- --- aat aat act agc tat ggt aac tat aca ttg ata

HV1ND gtg cca ata --- --- --- --- --- gac --- --- aat --- --- --- --- --- aat aat agg --- --- --- --- --- acc aat agt act aat tat --- --- --- agg tta ata

HV1EL gta cca ata --- --- --- --- --- gac --- --- aat --- --- --- --- --- gat agt agt --- --- --- --- --- acc aat agt acc aat tat --- --- --- agg tta ata

HV1Z84 gta cca ata --- --- --- --- --- gat --- --- gat --- --- --- --- --- gat aat agt gct aat acc agt aat acc aat tat acc aat tat --- --- --- aga tta ata

HV1MA gta caa ata --- --- --- --- --- gat --- --- gat --- --- --- --- agt gat aat agt --- --- --- --- --- --- --- --- --- agt tat --- --- --- agg cta ata

HV1ZH gta cca att ggg gga --- --- --- aat agt agt aat --- --- --- --- ggt gat agt agt --- --- --- --- --- --- --- --- --- aaa tat --- --- --- aga cta ata

SIVCZ gta aac cta ggg --- --- --- --- --- --- --- aat gag --- --- --- aac aac --- --- --- --- --- --- --- --- --- --- --- aca tat --- --- --- agg ata att

2B

DNA+pro

C-AF223955

C-AF223956

G-AF160501

G-AF405706

B-AB010291

B-AB010292

D-AF043594

D-AF090452

E-DQ060822

E-DQ060823

H-AY090454

H-AY090457

F-DQ899142

F-X69798

99

97

100

100

100

100

100

93

27

27

25

ClustalX2 Codon alignment

C-AF223955

C-AF223956

B-AB010291

B-AB010292

D-AF043594

D-AF090452

E-DQ060822

E-DQ060823

G-AF160501

G-AF405706

H-AY090454

H-AY090457

F-DQ899142

F-X69798100

100

100

100

100

100

100

99

48

21

38

E-DQ060822

E-DQ060823

D-AF043594

D-AF090452

B-AB010291

B-AB010292

C-AF223955

C-AF223956

G-AF160501

G-AF405706

H-AY090454

H-AY090457

F-DQ899142

F-X69798

100

100

100

100

100

100

100

100

46

43

22

Phylogeny of HBV S Protein

ClustalX2：Brazil/Nicaragua=> Angola =>Germany/Spain =>Japan => USA =>Vietnam

Brazil

Nicaragua

Angola

Spain

German

JapanUSA

Vietnam

1 2

3

4

5

C-AF223955

C-AF223956

G-AF160501

G-AF405706

B-AB010291

B-AB010292

D-AF043594

D-AF090452

E-DQ060822

E-DQ060823

H-AY090454

H-AY090457

F-DQ899142

F-X69798

99

97

100

100

100

100

100

93

27

27

25

Brazil

Nicaragua

Angola

Spain

German

JapanUSA

Vietnam

Condon alignment：Brazil/Nicaragua=>USA=>Angola=>Germany/Spain =>Japan=>Vietnam

12

3

4

5

C-AF223955

C-AF223956

B-AB010291

B-AB010292

D-AF043594

D-AF090452

E-DQ060822

E-DQ060823

G-AF160501

G-AF405706

H-AY090454

H-AY090457

F-DQ899142

F-X69798100

100

100

100

100

100

100

99

48

21

38

DNA+pro： Brazil/Nicaragua=> USA =>Vietnam / Angola /Japan/Germany/Spain

1

E-DQ060822

E-DQ060823

D-AF043594

D-AF090452

B-AB010291

B-AB010292

C-AF223955

C-AF223956

G-AF160501

G-AF405706

H-AY090454

H-AY090457

F-DQ899142

F-X69798

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polymerase-endonuclease amplification reaction (pear) and its potential applications

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