2

PCR(polymerase chain reaction) DNA RNA

.

,

, , , , ,

.

PCR,

cycle, ( DNA, dNTP, Mg2+),

primer , DNA polymerase .

(DMSO, , )

.

PCR?

PCR?

>>3

1. PCR

( 1).

(1) DNA(Denaturation)

DNA 94 15~30

DNA .

DNA polymerase

.

(2) Primer Annealing

DNA primer

2 primer

DNA annealing. Annealing

primer

55 30~1 annealing

. Annealing primer- DNA

mismatch .

primer mismatch primer

37~45annealing .

(3) (Extension)

4 (dNTP) , DNA

polymerase primer .

DNA , ,

Thermus

aquaticus(Taq) polymerase 72

30~10(10 kbp).Taq polymerase

DNA ( 60 nucleotides/sec, 70)1)

.

primer annealing ,

shuttle PCR.

) 94 30 ()

60~68 1~10(annealing, )

25~40 cycles

Shuttle PCR

annealing PCR

. (5 kbp

) shuttle PCR.

(4) Cycle

PCR n cycle 2n

. DNA

polymerase (DNA)

/ , annealing

primer anneal ,

1 PCR

DNA 2 primer tube . DNA polymerase DNA DNA .

cycle 0

DNA DNA

primer

primer (DNA )

DNA primer

primer (DNA )

DNA primer

primer (DNA )

DNA

cycle 1

cycle 2

cycle 3

cycle 4~30

PCR?

>>4

Pyphosphate

. 25~35 cycles

primer 2

DNA

. cycle

.

DNA cycle

. 1 copy PCR

. cycle

nested PCR . 1 PCR

primer

primer 2 PCR .

random primer DNA

primer extension preamplification(PEP)

2). DNA

primer(9~15 mer)

DNA

1 primer PCR

.

2.

(1) DNA Polymerase

DNA polymerase

. TaKaRa Taq DNA

70~80 60

nucleotides /. 92.5 130

, 95 40, 97.5 10.

(2) Mg2+

PCR 1.5~2.5 mM Mg2+

. Mg2+ PCR

. ,

primer annealing, DNA ,

primer-dimer . (

EDTA) Mg2+

DNA turn over

. Mg2+ dNTP

Mg2+

dNTP .

Mg2+ DNA

DNA , Taq

polymerase 10 mM MgCl2 40~50%

MgCl2

.

(3) dNTP

PCR, 20~200 M

. DNA polymerase dNTP

mismatch dNTP

3,4). dNTP PCR

dNTP misprimming

PCR 5). Mg2+

dNTP.

(4) DNA, RNA

PCR DNA RNA

. DNA

PCR

6).

PCR 7),

PCR 8,9).

DNA

PCR 10).

PCR 11).

DNA 1 PCR 105~106

3105

human genome DNA 1 , DNA

10 ng, DNA 1 ng .

nested PCR, PEP

1 DNA PCR . RNA

cDNA PCR

RT-PCR(Reverse Transcription-

Polymerase Chain Reaction) .

mRNA mRNA

, . RT-PCR

,

cDNA cloning . RT-

PCR Catrimox-14

RNA DNA

PCR?

>>5

. RNA

, Catrimox-14 ribonuclease

,14) ,15)

RNA .

(5)

primer set 1

tube PCR

PCR(multiplex PCR) .

18 primer

16). multiplex PCR

10%(v/v) dimethylsulfoxide(DMSO) 17). Taq polymerase

multiplex PCR.

Taq polymerase SDS(

PCR DNA polymerase

DNA polymerase PCR

. PCR Thermus aquaticus

DNA polymerase(Taq polymerase) polymerase

, .

PCRDNA polymerase

PCRDNA polymerase 94

, Hoffmann-LaRoche.

,

Taq polymerase. PCR

license.

DNA polymerase

>>6

DNA polymerase

>>7

1. DNA PolymeraseDNA polymerase

. , DNA

polymerase

.

DNA polymerase

Pol family, DNA polymerase

-family . 50

DNA polymerase family

A, B, C, X 4 .

family A B , C

DNA polymerase , X DNA

polymerase Terminal transferase .

family

, DNA ,

process DNA

nucleotide , -primer

.

, PCR DNA polymerase

DNA polymerase .

DNA polymerase 1

.

.

Pol family -

family . Taq polymerase Tth polymerase

Pol DNA

PCR

(mismatching) 35exonuclease

. , -family

Pfu polymerase Vent polymerase 35

exonuclease DNA

.

exonuclease

Pol

Family

Bacillus caldotenax BcaBEST DNA polymerase[TaKaRa] 53exo Bacillus stearothermophilus Bst DNA polymerase[BioRad] 53exo Thermus aquaticus TaKaRa Taq[TaKaRa]a) 53exo

Ampli Taq[Perkin Elmer]a) Stoffel fragment[Perkin Elmer]Taq DNA polymerase[ ] Gene Taq, Taq[Amersham]

Pol I Thermus thermophilus Tth DNA polymerase[Toyobo ]a) , single tube RT-PCRrTth DNA polymerase[Perkin Elmer]

53exo Tth DNA polymerse .

Thermus flavus Tfl DNA polymerase[Promega] Thermus ubiquitos Hot Tub DNA polymerase[Amersham]a)

Thermotoga maritima Ultma DNA polymerase[Perkin Elmer]a) 35exo .53exo .

Pyrococcus furiosus Pfu DNA polymerase[Stratagene]a) 35exo .

Pyrococcus sp.(GB-D) Deep Vent DNA polymerase[NEB] Thermococcus litoralis Vent DNA polymerase[NEB]

Tli DNA polymerase[Promega]Pyrococcus woesei Pwo DNA polymerase[B.M.]a)

TaKaRa Ex Taq[TaKaRa]a)

TaKaRa LA Taq[TaKaRa]a)

rTth DNA polymerase XL[Perkin Elmer]a)

Taq Plus DNA polymerase[Stratagene]a) LA-PCR

ELONGASE Amplification System[GibucoBRL]a)

Expand Long Template PCR System[B.M.]a)

a) PCR

DNA polymerase

>>8

. Pol 35exonuclease

Thermotoga

maritima Ultma polymerase

exonuclease .

PCR

. 1 BcaBEST polymerase

Bst polymerase PCR

().

2.

(1)

DNA polymerase

. Taq

polymerase 92.5

160 80% . 96

35 , 96 1, 55 1, 72

1 40 cycles .

PCR . Taq polymerase

Thermus aquaticus 80

Pfu polymerase Vent

polymerase Pyrococus furiosus

Thermococcus litoralis 100

.

Pfu polymerase 95, 60

95% , Vent Deep Vent

polymerase 95 6.7, 23

. BcaBEST Bst polymerase

Bacillus 75

30 .

PCR .

BcaBEST polymerase primer

( ), dideoxy sequencing

.

primer . DNA

DNA polymerase

85 -primer

. PCR primer annealing

DNA polymerase

70~75.

(2) DNA

Pol DNA

. M13 phage DNA primer

annealing DNA polymerase

. Pol Taq polymerase(TaKaRa)

BcaBEST polymerase(TaKaRa) Pfu

polymerase(Stratagene) DNA -

primer (0.6

U) primer

( 1) . BcaBEST, Taq

Pfu polymerase . 8 kb

M13 DNA BcaBEST, Taq 5

Pfu 20 .

PCR.

M B T P B T P B T P B T P M

2 5 10 20

(kb)

9.4

6.5

4.3

2.3

2.0

1 DNA polymerase primer

M13 DNA 45 primer annealing. -primer - 22 [32-P]dCTP dNTP0.6 U DNA polymerase, 2, 5, 10, 20 EDTA . BcaBEST65, Taq Pfu 72 . alkali agarose gel autoradiography . B: BcaBEST polymerase T: Taq polymerase P: Pfu polymerase

DNA polymerase

>>9

(3) (fidelity)

DNA polymerase DNA

. Taq polymerase

1~210-41,15,16) 1 DNA

10,000 nucleotide 1~2

DNA mismatch

. DNA polymerase

,

. , dNTP , -primer ,

, ,

. DNA polymerase

. 2

. 35

exonuclease -family Pfu polymerase

Vent polymerase Taq polymerase

. PCR DNA ,

cloning

( 2 kbp ),

.

PCR

reading-error ,

.

(mutagenic) PCRDNA polymerase

. Taq polymerase

PCR protocol

17,18). mismatch

MgCl2 , DNA

polymerase MnCl2

. DNA

polymerasePCR.

(4) Terminal extension

DNA polymerase DNA

, DNA polymerase DNA

3 1 nucleotide

19,20)( 2). DNA polymerase

terminal transferase

DNA polymerase

, DNA polymerase DNA

. PCRDNA

plasmid vector

. 3 1

nucleotide

, PCR 1 nucleotide

, DNA polymerase

primer . ,

PCR

. 3

nucleotide deoxyadenosine T

dideoxythymidine3open

circular. 35exonuclease

DNA polymerase

cloning

21,22). multicloning

site 0.5 kbp Haemophilus influenzae DNA

pUC plasmid

primer PCR, cloning

DNA polymerase

23). PCR T4 DNA

polymerase , T4 P. N. kinase 5-

, (A), T

(B)( 2)

3. Pfu polymerase

T .

PCR polyacrylamide gel

1 nucleotide Taq, Ex Taq(

DNA polymerase

Taq 2.0 10-4 40

Vent 6.6 10-5

Taq 7.2 10-5 40

Vent 4.5 10-5

Taq 8.9 10-5 41

Vent 2.4 10-5

Taq 2 10-5 8

Pfu 2 10-6

Tfl 2.1 10-4

Vent 3 10-5 42

Vent(exo) 4.4 10-4

2 DNA polymerase

DNA polymerase

>>10

), Pfu 60~70%, 50~60%,

30~40%.

PCR product SSCP

(single-stranded conformation polymorphism), DGGE

(denatured gradient gel electrophoresis)

microsatellite

.

Taq polymerase

Pfu polymerase

.

.

(5)

Pol

24,25), PCR Taq polymerase

mRNA PCR

26~28). (1~5

) RNA RNA

southern hybridization

. Tth polymerase

single tube RT-PCR(

PCR) . Tth polymerase

Mn2+ MnCl2

, EGTA

MgCl2 PCR

. single tube RT-PCR ,

,

2 RT-PCR.

Taq polymerase Tth polymerase

(88% identity)29,30)

DNA polymerase

.

(6)

PCR

DNA polymerase .

contamination dTTP

dUTP PCR , uracil-

N-glycosidase DNA ,

oligonucleotide

DNA dUTP 32),

hybridization probe

biotin dUTP

. GC

dGTP d7cGTP(7-DEAZA-guanosine

triphosphate) .

A, G, T, C nucleotide

DNA , Taq polymerase

. Vent polymerase Pfu polymerase

DNA .

Pol

pol. (%)

A Taq 400 37 91( cloning) Pfu 550 32 94

B Taq 57 21 73(T- cloning) Pfu 29 32 47

DNA cloning cloning

3 PCR cloning

Taq pfu polymerase PCR 250 ng((AA)) (TaKaRa blunting Kit), Hinc - pUC11850 ng (TaKaRa Ligation Kit Ver.2).((BB)) 50 ng pT7Blue(Novagen) . 21 10 JM109 , 1 200 plating . -galactosidase colorselection . colony plasmid .

A

Primer

5 -

B

DNA DNA polymerasedNTP

2 DNA polymerase

DNA polymerase primer DNA 1 nucleotide .

DNA polymerase

>>11

. nucleotide primer

G I() .

primer

, primer

.

.

. Taq

polymerase PCR

Pfu polymerase Vent polymerase

. 35exonuclease

mismatch . 3

5exonuclease Ultma polymerase

35).

(7) LONG PCR

PCR DNA

,

PCR

DNA .

PCR kbp

. DNA polymerase

, 2 kbp . -primer

10 kbp

, DNA

. 94, Barnes Pol

PCR

. Taq

polymerase PCR

35exonuclease

3 mismatch

35 exonuclease

Pfu polymerase Vent polymerase

PCR ,

, 10 kbp DNA

(

).

, TaKaRa Ex Taq polymerase Taq

polymerase

(). Ex Taq 35exonuclease

Taq 4

. DNA

kit TaKaRa LA(long & accurate) PCR kit

.

3. PCR PCR

DNA

Marker

Ultma

Deep VentVentPfuEx Taq

TaqUltm

aDeep VentVentPfuEx Taq

TaqMarker

0.5 kbp 1.0 kbp

Marker

Pfu

Ex Taq

Taq

Pfu

Ex Taq

Taq

Marker

4.0 kbp2.0 kbp

4.870

2.0161.3601.107926658489267

Marker

Ex Taq

Taq

Ex Taq

Taq

Marker

8.0 kbp6.0 kbp

4.870

2.0161.3601.107926658

19.3297.743 6.2234.2543.1422.6901.8821.489

3 DNA polymerase PCR

-phage DNA 5 ng primer 10 pmol, dNTP 10 nmol 50 PCR. DNA polymerase 2.5 Unit buffer . 94 0.5, 55 0.5, 72 0.5 25 cycles , 4 1% agarose gel.

DNA polymerase

>>12

-phage DNA

DNA

polymerase buffer

37).

Pol Taq polymerase(TaKaRa)Ultma

polymerase(Perkin Elmer) Pfu(Stratagene),

Vent(NEB), Deep Vent polymerase(NEB)

TaKaRa Ex Taq -phage DNA

0.5~8 kb PCR

agarose gel ( 3).

, Deep Vent, Vent, Pfu

. Pfu

polymerase 4 kb .

Taq Ex Taq 8 kb

. Taq

15 kb Ex Taq 4

40kbp . DNA

.

,

DNA

. Mg2+ (-

primer DNA dNTP

Mg2+ )

buffer MgCl2 , tube

Mg.

DNA

polymerase PCR Taq polymerase

, .

Taq polymerase

PCR.

PCR

. PCR Hoffman La Roche

PCR ,

,

. DNA polymerase Pol

DNA. ,

Super LA-PCR

. subunit

PCR

. DNA polymerase

, 14,38,39).

,

DNA polymerase

. ,

DNA polymerase

.

Marker-2

Marker-140 kbp38 kbp28 kbp20 kbp15 kbp12 kbp10 kbp8 kbp6 kbp

Marker-1

(kbp)(kbp)

24.822.619.417.015.012.2

8.6

27.523.1

9.4

6.6

4 Ex Taq polymerase long PCR

3 2.5 unit Ex Taq polymerase , 94 1 98 10, 68 20 30 cycles. 4 0.4% agarose gel .

DNA polymerase

>>13

1) Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf,

S. J., Higuchi, R., Horn, G. T., Mullis, K. B.,

Erlich, H. A.: Science, 223399, 487-491(1988)

2) Chien, A., Edgar, D. B., Trela, J. M. : J.

Bacteriol., 112277, 1550-1557(1976)

3) Wong, S. W., Wahl, A. F., Yuan, P.-M., Arai,

N., Pearson, B. E., Arai, K., Korn, D.,

Hunkapiller, M. W., Wang, T. S.-F., : EMBO

J., 77, 37-47(1988)

4) Wang, T. S.-F., Wong, S. W., Korn, D. : FASEB

J., 33, 14-21(1989)

5) Ito, J., Braithwaite, D. K. : Nucl. Acids Res.,

1199, 4045-4057(1991)

6) Braithwaite, D. K., Ito, J. : Nucl. Acids Res.,

2211, 787-802(1993)

7) Myers, T. W., Gelfand, D. H. : Biochemistry,

3300, 7661-7666(1991)

8) Lundberg, K. S., Shoemaker, D. D., Adams, M.

W. W., Short, J. M., Sorge, J. A., Mathur, E. J. :

Gene, 110088, 1-6(1991)

9) Kong, H., Kucera, R. B., Jack, W. E. : J.

Biol. Chem., 226688, 1965-1975(1993)

10) Uemori, T., Ishino, Y., Fujita, K., Asada, K., Kato,

I.: J. Biochem., 111133, 401-410(1993)

11) Koboev, O. K., Luchkina, L. A., Akhmedov, A.

T., Bekker, M. L. : J. Bacteriol., 114455, 21-

26(1981)

12) Ishino, Y., Ueno, T., Miyagi, M., Uemori,

T., Imamura, M., Tsunasawa, S., Kata, I. : J.

Biochem,111166, 1019-1024(1993)

13) Ishino, Y. : Am. Biotechnol. Lab., 1100, 47(1992)

. PCR Primer

. PCR Primer

. PCR Primer ,

PCR Primer

>>14

. PCR Primer

PCR primer 2

(single strand)DNA .

Primer PCR

. Primer

1) , 2) primer , 3) GC , 4)

primer 2, 5) Tm , 6)

, primer .

PCR primer 2

DNA . Primer DNA

. primer

primer PCR

PCR . DNA

(5) sense strand(), (3)

antisense strand() primer .

primer primer

OligoTM .

1. Primer

(1) Primer

Primer 15~30 20~25

. primer DNA

annealing . LA PCR(long

and accurate PCR) 30 primer

.

(2) Primer

2 primer annealing. 3

primer primer

dimer .

(3) GC

GC 50% GC

AT-rich . primer 3

DNA primer 3 AT-

rich .

(4) Primer 2

Primer 2

.

(5) Tm

2 primer Tm .

primer Tm

. Annealing primer Tm

55~65

. Annealing mispriming

PCR .

(6)

Primer0.2~1 M.

2. Primer primer

.

OligoTM, GENETYX OligoTM

.

(1) OligoTM

N.B.I.(Natonal Bioscience, Inc.)

OligoTM PCR, sequencing,

, hybridization primer probe

oligonucleotide .

OligoTM Tm , G

primer .

(2) PCR Primer

OligoTM primer

. PCR primer

oligonucleotide.

Tm oligonucleotide Tm

25~75% .

1 Tm plot OligoTM

.

3 , 5

PCR Primer

>>15

PCR Primer

>>16

Primer DNA DNA

(duplex stability) .

PCR primer 3 DNA

primer

primer 3 G

.

Dimer hair pin

3(stem)

primer .

3

DNA 3 7

primer .

5 primer

. annealing

. 2 PCR

. , annealing , primer

, Tm , GC .

primer PCR primer

.

. PCR Primer

PCR primer .

PCR primer

,

.

primer

.

homology primer

(hybridizability)

.

PCR

(thermal cycler), ,

oligonucleotide primer

2 PCR primer .

oligonucleotide

1 OligoTM

2 PCR

PCR Primer

>>17

,

pair .

PCR

primer

.

oligonucleotide probe primer

.

homology

.

(HYB simulatorTM)5, 6) . probe

primer PCR

. PCR primer

.

1. PCR Primer

(1)

Primer target primer

. DNA DNA

nucleotide Adenine(A) Thymine(T),

Guanine(G) Cytosine(C)

2 , 3

, A T primer G

C primer . Biotin,

Avidin , DNA

mismatch

.

. PCR 2

primer 1 primer

target 2 primer

1 Tm

38 oligonucleotide Tm , AT pair 2, GC pair 4 (a), Tm=81.5 + 0.41(%GC)-600/L (L:probe ) (b), Breslauer nearest neighbor (c), Santa Lucia nearest neighbor (d) Tm .

PCR Primer

>>18

target ,

mismatch

.

Oligonucleotide melting

temperature(Tm). Tm oligonucleotide

DNA hybridize

hybridization 50% oligotide hybridize

50% DNA .

.

AT pair 2, GC pair 4

oligonucleotide Tm .

( 1a).

Bolton

Tm=81.5+0.41(%GC)-600/L

L nucleotide

( 1b).

G A C

(nearest neighbor: nn)

.8) nn

(entrophy- S entrophy- H)

, Tm.

Tm= H/( S+RIn(c)-273.15+16.6 log[Na+])

R: (1.987 cal//mol)

c: oligonucleotide

[Na+]: 1

Mg Na [Na+]=4[Mg2+]1/2

Tm

( 1c).

Wayne State Santa Lucia

nn

data ( 1d)

.

Primer Tm PCR annealing

?

background .

Tm 50% oligonucleotide hybridize

50% oligonucleotide

Tm annealing

.

mismatchhybridization

. Taqpolymerase

mismatch primer DNA

. PCR

annealing Tm

(5~15) .

(2)

Primer

homology 2, 4) . homology

homology oligonucleotide

( 2).

homology

(hybridizability) .

primer GenBank

database

mismatch nn

primer (Tm

G) .

parameter .

target Tm cross hybridization

Tm primer

target (

3a). Tm cross hybridization

2 10 mer primer homology

Primer 2 primer 1 homology 70% GC .

PCR Primer

>>19

( 3b). database

.

primer

. PCR primer 5 3

DNA 3 GC

3 Tm primer

3 target DNA

. 3

.

3 GC .

database 5 mer 6 mer

primer 3

.

PCR .

(3) 2

PCR primer

primer hair pin

false positive

. PCR

hair pin

hair pin .

PCR primer target

3 primer

Primer target () ( G) HYBsimulator . (a) target G cross hybrization G . . (b) target G 5 kcal/ml cross hybrization . .

(a)

kcal/m

ol

Oligonucleotide

(b)

Oligonucleotide

PCR Primer

>>20

primer hair pin PCR

.

Oligonucleotide hair pin

kcal/mol . 4(a) target

Tm

oligonucleotide hair pin primer

hair pin

. hair pin

. DNA cDNA

DNA primer hybridize

DNA hair pin primer

hybridization . Primer hybridization

hair pin DNA

. primer

hybridizatoin hair pin

. primer

primer

hair pin

primer hybridizatoin .

hair pin.

(4) Primer

PCR primer

primer

hybridize primer

dimer target hybridization

. primer dimer sense, antisense

sense antisense 3 . sense

antisense hybridize

, 5-AGCT-3 (3-TCGA-5)

dimer. dimer

(4b).

Oligonucleotide

Oligonucleotide

4 Primer hair pin dimer

Primer target () (G) hair pin (Kcal/mol) (a) dimer (Kcal/mol) (b) HYB simulator . ( ) hair pin dimer

PCR Primer

(5)

Cloning

PCR

. PCR

PCR

primer .

GC PCR

know-how .

GC GC

primer

.

PCR PCR

. PCR

. 1000 bp

know-how(long PCR ) .

primer dimer

agarose gel

acrylamide gel

.

(6) Primer

10 mer 40 mer

primer .

sense antisense Tm

.

primer Tm

annealing mismatching

hybridization .

mismatching .

human genome 17

mer(4 17 ) human

. PCR 2

primer 17 mer

20 mer primer

.

2. PCRPrimer

(1) Nested PCR

Nested PCR primer primer

2 PCR . primer

PCR , DNA primer

primer . 2 PCR

primer primer 4

primer .

(2) Multiplex PCR

Multiplex PCR primer

,

. primer

primer dimer primer

. PCR agarose gel

.

(3) Long PCR

PCR 1,000 bp primer

PCR .

primer 3 PCR

DNA PCR

. PCR .

(4) Competitive PCR

primer target

PCR PCR

target

. primer

cross hybridization.

(5) PCR Cloning

PCR subcloning

primer 5

PCR .

primer

hair pin dimer

.

(6) PCR

primer 3 hybridization

mismatch primer

5

>>21

PCR Primer

>>22

. 3 mismatch DNA

primer.

mismatch sense primer

antisense primer .

primer mismatch Tm

sense antisense

. 3 GC

3 mismatch 3 DNA

hybridization .

3 GC primer mismatch

. 3 hybridization

3 2 mismatch

. genome

100%(Home), 50%(Hetero), 0%()

.

. mismatch hybridization

primer(10-20 mer)

.

(7) Primer

family primer

PCR family

. type mycobacteria

mycobacteria

primer PCR .

family cloning

. family

database .

database , mycobacteria

(mycobacteria database ),

bacteria (GenBank bacteria

database ),

(GenBank primate database )

primer .

. PCR Primer ,

agarose acrylamide

.

oligonucleotide HPLC(High performance

liquid chromatography)

OD oligonucleotide .

(oligonucleotide purification

catridge column: OPC).

oligonucleotide catridge

.

nucleotide capillary gel

.

(1) Oligonucleotide

oligonucleotide

. PCR , primer pmol

. 1 ml

oligonucleotide 260 nm

OD260(optical density) units/ml .

A=cl(A:, : mol, c: mol

(mmol/ml), l: (1 cm)) c=A/l=A/

mol . 1 mol

10,000 primer

. oligonucleotide 1 OD260 unit

33 .

1 nucleotide

: DMTr~40 mer, column : Capcell Pak SG-300(6.0300 mm), :(A) 5% CH3CN 0.1 M TEAA buffer, : 30%(B)~80% (B) (20), : 1.0 ml/, : 40, : UV(254 nm)

PCR Primer

>>23

(2) HPLC

Oligonucleotide HPLC

column HPLC column.

A. Chromatography(RP-HPLC)

silicagel

oligonucleotide 18

octadecyl 5

100~300 octadecylan(ODS)

.

column

oligonucleotide 5

DMTr

capping 5 free

nucleotide .

nucleotide ( 1)

HPLC DMTr 80% acetate

HPLC.

, -

acetonitryl, -methanol pH

tetraethyl ammonium acetate(TEAA)

. tetraethyl DNA

DNA column

.

cylan

(FluofixTM[NEOS]) .

,

oligonucleotide column

( 2).

B. Chromatography(DEAE-HPLC)

chromatography

. DNA -

. , chromatography

oligonucleotide

.

oligonucleotide

.

DNA Diethylaminoethyl(DEAE)

.

TSK-gel DEAE-NPR(TOSO)

. oligonucleotide

acetonitryl

formic acid form

NaCl

oligonucleotide .

2 FluofixTM

: 28 mer CGAATTCGAGCTCGAGATGAAGCTCTTT, column: FLUOFIX300N(4.6 150 mm), : (A) 50mM ammonium acetate (B)(A)/MeCN=50/50, : 0% (B) ~10% (B) (30), : 1.0 ml/, : , : UV(254 nm)

3 TSK-gel DEAE-NPR

: TSK-gel DEAE-2 SW(4.6250 mm). (A) 20% CH3CN 0.2 MHCOONH4 (B) 20% CH3CN 0.1 M HCOONH4 : 30% (B) ~70% (B)(20), : 0.8 ml/, : 40, : UV(254 nm)

PCR Primer

>>24

, , cartridge

Sephadex G-25 (NAP-10[Pharmacia])

.

,

cartridge .

C. Cartridge (Oligo-PakTM[Perseptive],OPCTM[Perkin-Elmer], Sep-Pak[Waters] )

Octadecysilan(ODS) cartridge

HPLC .

.

. cartridge

apply

cartridge . 1

. Flash

cartridge

. , 0.1 M TEAA 0.05 M

TEAB(tetraethyl )

. DMTr oligonucleotide

.

cartridge 10 OD

Unit oligonucleotide .

HPLC . PCR

primer cartridge

.

D. Capillary Gel

nucleotide polyacrylamide gel

capillary gel

. Capillary tube polyacrylamide gel

1

. capillary acrylamide

gel .

capillary polymer tube

tube

.

1) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K.

B., Horn, G. T., Erlich, H. A., Arnheim, N. :

Science, 223300, 1350-1354(1985)

2) Wilbur, W. J., Lipman, D. J. : Proc. Natl. Acad.

Sci. USA, 8800, 726-730(1983)

3) Smith, T. F., Waterman, M. S. : Adv. Appl. Math.,

22. 482-489(1981)

4) Pearson, W. R., Lipman, D. J. : Proc. Natl. Acad.

Sci. USA, 8855, 2444-2448(1988)

5) Mitsuhashi, M., Cooper, A., Ogura, M.,

Shinagawa, T., Yano, K., Hosokawa, T. :

Nature, 336677, 759-761(1994)

6) Mitsuhashi, M., Hosakawa, T. : , 5522,

530-541(1994)

7) Bolton, E. T., McCarthy, B. J. : Proc. Natl. Acad.

Sci. USA, 4488, 1390(1962)

8) Breslauer, K. J., Frank, R., Blocker, H., Marky,

L. : Proc.Natl. Acad. Sci. USA, 8833, 3746-

3750(1986)

9) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K.

B., Horn, G. T., Erlich, H. A., Arnheim, N. :

Acetonitryl 5 ml catridge

flash

5 ml acetonitryl

flash

10 ml.

Ammonia apply .

10 ml.

25% methanol/ 10 ml nucleotide

flash

10 ml methanol

2% Trifluoreacetic acid(TFA) 5 ml 2

Oligonucleotide DMTr

flash

10 ml TFA

flash

30% methanol/3 mlnucleotide

PCR Primer

>>25

Science, 3377,170-172(1985)

10) Tanaka, T., Letsinger, R. L. : Nucl. Acids Res.,

1100, 3249-3260(1982)

11) Ikehara M. , , 2266, 531 537(1981)

12) Michelson, A. M., Todd, A. R. : J. Chem. Soc.,

2632(1955)

13) Khorana, H. G. : Pure. Appl. Chem., 1177, 349-

381(1968)

14) Letsinger, R. L., Ogilvie, K. K. : J. Am.

Chem. Soc., 8899, 4801-4803(1967)

15) Letsinger, R. L., Lunsfold, W. B. : J. Am.

Chem. Soc., 9988, 3655-3661(1976)

16) Matteucci, M. D., Caaruthers, M. H. : J. Am.

Chem.Soc., 110033, 3185-3191(1981)

17) Sinha, N. D., Biernat, J., McManus, J.,

Koster, H. : Nucl. Acids Res., 1122, 4539-

4557(1984)

18) Froehler, B. C., Matteucci, M. D. :

Tetrahedron Lett., 2277, 467-472(1986)

19) Adams, S. P., Kavka, K. S., Wykes, E. J.,

Holder, S. B., Galluppi, G. R. : J. Am. Chem.

Soc., 110055, 661-663(1983)

20) Schaller, H., Weimann, G., Lerch, B., Khorana,

H. G. : J. Am. Chem. Soc., 8855, 3821(1963)

21) Buchi, H., Khorana, H. G. : J. Mol. Biol., 7722,

251(1972)

22) Smith, M., Rammler, D. H., Goldberg, I. H.,

Khorana, H. G. : J. Am. Chem. Soc., 8844,

430(1962)

23) Fritz, H. J., Belagaje, R., Brown, E. L., Fritz,

R. H., Jones, R. A., Lee, R. G., Khorana, h. G. :

Biochemistry, 1177, 1257-1267(1978)

24) Gait, M. J., Matthes, H. W. D., Singh, M.,

Sproat, B. S., Titmas, R. C. : Nucl. Acids Res.,

1100, 6243(1982)

25) Dahl, B. H., Nielsen, J., Dahl, O. : Nucl. Acids

Res., 1155, 1729-1743(1987)

26) Hayakawa, Y., Uchiyama, M., Noyori, R. :

Tetrahedron Lett., 2277, 4191-4194(1986)

27) Kato, K : II I .

pp147-171, (1991)

28) Newton, C. R., Greene, A. R., Heathcliffe, G.

R., Atkinson, T. C., Holland, D., Markham, A.

F., Edge, M. D. : Anal. Biochem., 112299, 22-

30(1983)

>>26

. DNA

. DNA

.

PCRDNA

PCRDNA

>>27

. DNA

PCR DNA

DNA,

. DNA

PCR DNA .

DNA

,

.

DNA

.

DNA , library

DNA proteinase

K/phenol ,

PCR DNA .

DNA

. PCR DNA

DNA

.

1. Proteinase K/Phenol 1, 2)

(1)

SDS , ribonuclease RNA

. proteinase K

phenol .

, DNA

.

(2)

buffer(10 mM Tris-HC1(pH8.0), 0.1 M

EDTA(pH 8.0), 20 /ml pancreas ribonuclease,

0.5% SDS)(10 mM EDTA DNA

)

20 mg/ml proteinase K (

, )

0.5 M Tris-HC1(pH8.0)-saturated phenol

10 M Ammonium acetate

Ethanol 70% ethanol

TE (10 mM Tris-HC1(pH8.0), 1 mM

EDTA)

(3) DNA

10 cm plate (107) , PBS

scraper plate

15 ml polypropylene tube .

41500g, 5

.

() pancreas

ribonucleasebuffer 2 ml,

37 1.

(4) DNA

(100 mg)

,

,

.

pancreas ribonuclease

buffer 4 ml , 15 ml polypropylene tube

37 1.

(5) DNA

(3) (4) 100

/ml 20 mg/ml proteinase K 1/20

,

50 3.

0.5 M Tris-HCl(pH8.0) phenol

.

50 mM Tris-HCl(pH8.0) -10 mM EDTA

, 50 ml polypropylene tube

phenol . Tip

. 5000g, 15

. phenol 3 .

DNA 15 ml 50 ml polypropylene tube

, 10 M ammonium acetate 0.2 vol.

. 2 vol. ethanol

. DNA

, DNA tip

PCRDNA

>>28

, 70% ethanol

ethanol .

2 . DNA

100~150 kb .

200 kb DNA

phenol 50 mM Tris-HC1(pH8.0)-10

mM EDTA(pH8.0) , ethanol

DNA.

DNA

5,000g 5

, 70% ethanol 2.

ethanol .

DNA .

1 ml TE

DNA .

TE.

260 nm , 1 OD260=50 /ml

. 107 40

, 100 mg 200 DNA

.

2. Proteinase K/Phenol/Chloroform 3, 4)

(1)

1 , PCR

.

(2)

1 PBS(3.56 g Na2HPO4 12H2O, 0.52 g

NaH2PO42H2O, 8.5 g NaCl/1).

2 buffer(20 mM Tris-HCl(pH8.0), 20 mM

EDTA, 300 mM NaCl, 0.4% SDS).

20 mg/ml Proteinase K (

, ).

0.5 M Tris-HCl(pH8.0)-saturated phenol/chloroform/

isoamyl alcohol(25 : 24 : 1).

TE (10 mM Tris-HCl(pH 8.0), 1 mM

EDTA).

(3) DNA

107 1 ml 1PBS, 2

buffer .

, 1PBS , 2

buffer.

vortex

.

100 /ml 20 mg/ml

proteinase K 1/20 vol. , vortex

50 3.

0.5 M Tris-HCl(pH8.0)-saturated

phenol/chloroform /isoamyl alcohol

tube .

5,000g, 15 .

Tip , DNA

tube chloroform

. 5,000g, 15 DNA

.

1/10 3 M sodium acetate ,

ethanol 2 , -20 1

.

5,000g, 5 DNA

, 70% ethanol 2.

DNA TE buffer . RNA

1 20 /ml

pancreas ribonuclease , 37 1

, .

DNA 20 /ml pancreas ribonuclease

37 1 , chloroform/isoamyl

alcohol .

260 nm 1 OD260=50 /ml

.

3. 3)

(1)

PCR DNA . DNA

, tube

DNA.

(2)

50 mM NaOH

Mineral oil(Sigma # M-5904)

1 M Tris-HCl(pH7.0)

(3) DNA

, 1.5 ml eppendorf tube,

freeze-thaw 2

.

5106 100 50 mM NaOH

.

vortex .

Spin-down 150 Mineral oil .

Heating block 95 10.

1 M Tris-HCl(pH7.0) . 100 50

mM NaOH 16 . Mineral oil

DNA

.

-70.

4. Boiling4)

(1)

PCR DNA

.

(2) DNA

eppendorf tube ,

. ,

. 100 mg 1 ml

.

Tube , Heating block

15DNA .

PCR

DNA . DNA

.

DNA

. DNA

PCR .

DNA PCR ,

.

,

, .

DNA

1/100 .

DNA,

, PCR .

. DNA

PCR ,

. ,

. , ,

, , , , , ,

. PCR

.

.

.

PCR

,

.

,

. ,

phenol/chloroform, guanidine thiocyanate

, .

.

1.

(1)

virus viral diarrhoea

PCRDNA

>>29

PCRDNA

>>30

enterovirus, . , viral diarrhoea

A rotavirus, adenovirus, calcivirus,

PBS10~20%

8,000 rpm, 10

phlorocarbon

12,000 rpm, 5

1% SDS, 1 mM EDTA, 100 Proteinase K,

10 mM Tris, 37, 1

95, 10(Proteinase K )

Phenol/chloroform 1, Chloroform 1

Ethanol , pellet TE buffer DDW

PBS10~20%

8,000 rpm, 10

phlorocarbon

12,000 rpm, 5

400 polyethylene glycol 6000(8%), NaCl(0.4 M)

12,000 rpm, 5

Pellet 150 DDW, 2 Proteinase Kbuffer*1 400 /mlProteinase K,

3730

CTAB(1.25%), NaCl(0.45 M),5630

Phenol/chloroform 1, Chloroform 1

Ethanol , pellet TE buffer DDW

PBS10~20%

8,000 rpm10

phlorocarbon

12,000 rpm5

250 320 6 M guanidine thiocyanate

RNaid martrix, 10

4,000 rpm1

, 400 buffer

6,000 rpm1

, 400 buffer

8,000 rpm1

, 400 buffer

12,000 rpm1

, pellet

50 DDW, 6510

12,000 rpm1

PCR

* 1 2Proteinase K buffer: 2% SDS, 25 mM, 200 mM Tris, 0.3 M NaCl

PCRDNA

>>31

norwalkvirus, astrovirus virus

PCR .

enterovirus PCR .

, ssRNA virus, dsRNA virus, dsDNA

virus PCR

.

ssRNA virus .

.

A rotavirus, calcivirus PCR

.

, PBS(phosphate buffered saline)

10~20% , 8,000 rpm 10

. phlorocarbon

, 12,000 rpm 5

PCR .

dsRNA

A rotavirus5)

ssRNA calcivirus6)

. phenol/chloroform

guanidine thiocyanate(GTC)

Kit(RNaid) ( 1,

2, 3), 1.5 ml micro tube

.

, A rotavirus phenol/chloroform

5) phlorocarbon 400

1% SDS, 0.1 mg proteinase K, 1mM

EDTA, 10 mM Tris(pH8) ,

37 1. 95 10 proteinase

K phenol/chloroform ,

chloroform . 2

cold ethanol sodium acetate ethanol

. PelletTE(Tris-EDTA) buffer

DDW(deionized distilled water) , PCR

( 1). 1.

, calcivirus phlorocarbon 400

1 PCR A rotavirus

M( marker), KU( 1, , 749), (), 179~507(), S2( 2, , 657), YO( 3, , 582),HO( 4, , 394), M( marker), 526~665(), M( marker), 8 6 , 1 1, 4 5 .

M KU(

1)

179

184

(

4)

402

(

1)

487

(

4)

507

(

4)

S2(

2)

YO(

3)

HO

(

4)

M 526

(

4)

660

(

4)

665

M

4870

2016

48926780

13601107926658

PCRDNA

>>32

8% polyethylene, 0.4 M

NaCl 12,000 rpm 10

. Pellet proteinase K buffer*1)

37 30 . 1.25 %

CTAB(cetyl-trumethyl ammonium bromide), 0.45 M

NaCl 56 30

. phenol/chloroform

ethanol ( 2). virus

, ssRNA

norwalkvirus (small round

structured virus) virus PCR6).

GTC ,

glass powder silica gel

kit

. kit , phenol

.

RNase

ssRNA

. , kit DDW buffer

DEPC(diethyl pyrocarbonate)

RNase free tube pipette tip

RNA virus PCR

( 3).

(2)

.

Parvovirus B19 ssDNA

, HHV-6 cytomegalovirus

buffy coat

.

Parvovirus B19 B

. 100

400 buffer[ 500 /ml proteinase K,

0.5% SDS, 5 mM EDTA(pH8), 10 mM Tris-

HCl(pH8)] , 50.

phenol/chloroform 2

chloroform 1 ethanol .

70 4

PCR .

phenol/chloroform , nested PCR

southern blotting

.

cross contamination .

PCR

7).

HHV-6 cytomegalovirus , Ficoll-paque

phenol/chloroform

.

.

8).

. 10 TE buffer

, 10,000 rpm 10

. pellet .

(3)

,

.

DTT(dithiothreitol) 1 N

NaOH ,

.

1 N NaOH 10

, 1 M NaH22PO4

10,000 rpm 10 . PBS 2

40~100 lysozyme

[1~2 mg/ml lysozyme: 10 mM Tris(pH8), 10 mM

EDTA, 0.5% SDS] , 37 30~2

proteinase K 200 /ml

37 .

, ,

phenol/ chloroform 9).

lysozyme . 0.1% DTT

, 10,000 rpm 10.

NaOH , pellet

(SDS, EDTA, proteinase K, lysozyme

)

.

.

PCRDNA

>>33

(4) , , ,

.

pellet , 10

ml 1 ml ,

PCR

.

phenol/chloroform kit

.

.

(5) ,

PBS , pellet

TE buffer PCR ,

phenol/chloroform kit

.

.

(6)

2,000 rpm, 30

.

pellet . Pellet

PBS 2 ,

. pellet

phenol/chloroform

kit .

PCR ,

.

,

. ,

ssRNA norwalk virus

. ,

DNA

Parvovirus B19

PCR cross

contamination . ,

HHV-6

,

phenol

. ,

.

.

.

, screening

DNA

.

,

DNA

.

,

,

.

screening

.

.

,

.

DNA .

1. 10)

( 1).

.

.

,

,

.

PCRDNA

>>34

. ,

,

.

.

,

.

1 5103~2105(

2104) PCR

DNA.

,

DNA

. MASA(mutant allele specific amplification)

DNA

11), PCR-SSCP

10~20%

. microsatellite marker

LOH(loss of heterozygosity)

50~60%

LOH . ,

DNA () ,

,

"negative fulse ()"

.

2. (, )

. , 1,500 rpm 5

.

. 1%

saponin.

,

slide glass

.

, PBS

.

1 DNA

(A), (B): DNA 0.8% agarose gel . Lane a DNA, lane b slide glass DNA, (C): p53 exon 5 PCR-SSCP (silver staining). Lane 2 band .

-Hind

III d

igest

X1

74-Hae

III digest

a b

(A) (B)

(C) 1 2 3 4

: , ,

: , , ,

: ,

: ,

:

: ,

: ,

:

: ,

: ,

1

PCRDNA

>>35

DNA . FISH(fluorescence in situ

hybridization)

,

FISH DNA

. ,

microtube , 2 carnoys

solution(methanol:acetate=3:1)

. 3,000~4,000 rpm 10

, carnoys

solution .

FISH , 1104~5

104 DNA *1. carnoys

solution acetate DNA

, PBS DNA

( 1A).

slide glass

DNA .

slide glass

. , DNA

( 1B), PCR

12).

3. DNA 13)

DNA

.

.

0.5 mlmicrotube 14,000

rpm 10 100

HMW*2-proteinase K(100 /ml).

37 4.

HMW-saturated phenol ,

.

3,000 rpm 10 tip

phenol

, 2.

3,000 rpm 10

chloroform/isoamylalcohol(24:1)

, 4.

3,000 rpm 10 ,

, 0.1 vol. 8 M ammonium acetate , 2

100% ethanol , 0.5 2 % glycogen

, -201ethanol .

4 14,000 rpm 20

glycogen DNA*3.

DNA 70% ethanol

, TE buffer(20~100 ) .

1 (A), (B) DNA

0.8% agarose gel . carnoys

solution

DNA . 1

(C)

DNA PCR-SSCP p53

.

*1 1104 10~200 ng DNA .

*2 10 mM Tris-HCl(pH 8.0), 150 mM NaCl, 10 mM EDTA-NaOH(pH8.0),0.1% SDS.

*3 DNA ethanol .

DNA

.

.

.

. DNA

DNA ,

.

pellet

proteinase K/phenol/chloroform DNA

. DNA ethanol

glycogen DNA

.

, ,

, DNA

.

tube

DNA PCR-SSCP

PCRDNA

>>36

K-ras codon 12 point

mutation 1, 2).

DNA .

1. *1

Proteinase K : Proteinase K(Merck)

1 mg/ml (10

mM Tris-HCl pH7.4, 10 mM EDTA, 0.15 M

NaCl, 0.4% SDS)*1.

SS-phenol

) Phenol(Wako, chromatography) (60

), 8-hydroxyquinoline

0.1% .

) 1 M Tris-HCl(pH8.0)*1

, .

) .

) ), ) 2~3.

v) 0.1 M Tris-HCl(pH8.0)*1

, .

) (

).

) 2-mercaptoethanol 0.2%

, 4.

Chloroform

3 M sodium acetate(pH7.4)*1

Glycogen(molecular biology grade: Boheringer

mannheim)

Cold ethanol(-20)

80% Cold ethanol

TE buffer(10 mM Tris-HCl pH7.4, 1 mM

EDTA)*1*1 , autoclave .

2.

(1)

, -80.

, .

(2)

, 2,500 rpm 4

10.

1 DNA UV spectacle 270 nm .

PCRDNA

>>37

tube

.

Pellet 2 ml ,

2,500 rpm 4 5.

.

, .

Pellet -80.

3. DNA 16)

pellet 1 ml proteinase K

( / 10

proteinaseK), 65 15

37.

1:1 phenol:chloroform mixed,

10,000 rpm 10.

tube1:1 phenol:chloroform

, 10,000 rpm10.

400 2 eppendorf tube

, 1/10 (40 ) 3 M sodium acetate 1

glycogen 2.5 (1 ml) cold ethanol

, -20 1 (

,

).

10,000 rpm 10 ,

.

80% cold ethanol (0.5~1 ml)

, 10,000 rpm 10.

, Speed Vac Concentrator (Savant

).

TE buffer 50 ,

DNA .

DNA

. , -

80

DNA .

pellet DNA

. Pellet , -20

PCR .

DNA

270 nm 260 nm/280

nm 1.6 ( 1).

, PCR (

2). 2 ml DNA

, DNA ng

. 5~6 PCR (

2). Ethanol glycogen carrier

DNA.

1) Blin, N., Stafford. D. W. : Nucl. Acids Res., 33,

2303 (1976)

2) Maniatis, T., Fritsch. E., F., Sambrook. J, : in

Molecular cloning : a laboratory handbook, pp.

9. 14-9, 19, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY (1982)

3) Rolfs, A., Schuller, I., Finckh, U., Weber Rolfs,

I. : in PCR : Clinical diagnostics and research,

pp. 79-89, Springer-Verlag. Berlin, Heidelberg

(1992)

4) Jackson, D. P., Hayden, J. D., Quirke, P. : in

PCR : A practical approach (ed. McPherson, M.

J., Quirke, P., Taylor, G. R.), pp. 29-33, IRL

Press, Oxford (1991)

2 DNA PCR , K-ras exon 1 .

MW marker, lane 1~8 DNA, lane 9 negative control.

MW 1 2 3 4 5 6 7 8 9

PCRDNA

>>38

5) Taniguchi. K., Wakasugi, F., Pongsuwanna, Y.,

Urasawa, T., Ukae, S., Chiba, S., Urasawa, S. :

Epidemiol. Infect., 110099, 303~312 (1992)

6) Jiang, X., Wang, J., Graham, D. Y., Estes, M.

K. : J. Clin. Microbiol., 3300, 2529~2534 (1992)

7) Frickhofen, N., Young, N. S.: J. Virol. Methods,

3355, 65~72 (1991)

8) Kondo, I. et al ., 3355, 3041~3047

(1990)

9) Narita, M., Matsuzono, Y., Shibata, M.,

Togashi, T. : Acta Pediatr., 8811, 997~1001

(1992)

10) Yatani, K. et al. :

, pp. 45-67, (1990)

11) Tskeda, S., Ichii, S., Nakamura, Y. : Human

Mutation, 22, 112-117(1993)

12)Wada, C., Shinoya, S., Fujino, Y., Rokuhiro, H.,

Akahoshi, T., Uchida, T., Ohtani, H. : Blood,

8833, 3449-3456 (1994)

13) :

protocol, pp, 16-19, (1983)

14)Kondo, H., Sugano. K., Fukayama. N.,

Kyogoku, A., Nose, H., Shimada, K., Ohkura,

H., Ohtsu, A., Yoshida, S., Shimosato, Y. :

Cancer, 7733. 1589-1594 (1994)

15) Fukayama, N., et al : . 4411,1017-

1023(1993)

16)Davis, L. G., Dibner. M. D., Battey. J. F. :

Basic Methods in Molecular Biology, pp. 44-46.

Elsevier, New York (1986)

TaKaRa Gradient ThermalCycler Dice

PCR, PCR!!

1. Compact & Stylish BodyControl Panel & Display wing-panel .

2. Pop-up Menu Programing .

3. & Annealing Gradient , Touch Down PCR

4. PC software update data backup PCR history error history .

>>39

. Agarose gel

. Polyacrylamide gel

. Dot hybridization

PCR

PCR

>>40

I. Agarose Gel

Agarose gel PCR

.

DNA DNA

. agarose

, PCR 500 bp

DNA agarose gel

. PCR

agarose

.

1. Agarose Gel

(1) Agarose

agarose gel .

agarose gel 0.5~20 kb DNA

. agarose

.

Agarose L03 (TaKaRa Code 5003) .

( 15~20 cm gel)

submarine DNA mini gel

.

(2) Agarose Gel 1)

A.

50TAE : 242 g Tris, 57.1 ml acetic acid, 100 ml

0.5 M EDTA(pH 8.0)1 .

1TAE : 50TAE 50 (:

40 mM Tris-acetate, 1 mM EDTA), 1

TAE 0.5TBE(45 mM Tris-

borate, 1 mM EDTA)

.

6l oading buffer: 0.25% bromophenol blue, 0.25%

Xylene cyanol FF, 30% glycerol, 10

mg/ml ethidium bromide.

B. Gel

1% agarose, 1 g agarose 100 ml 1TAE

. Gel

60

. GelDNA

1 .

gel .

Gel tray comb set, 3~5 mm

gel. Comb set , gel tray 1 mm

.

30gel.

C.

6loading buffer, well.

10~100 ng, 10 .

5 V/cm gel , mini gel

, 100 V.

D.

DNA interchelation,

ethidium bromide . 0.5 /

ethidium bromide(1TAE )

gel30.

Ethidium bromide gel , 0.5 /

. ethidium bromide

(-) gel

ethidium bromide

.

1TAE 15 band

. .

Gel 254 nmUV transilluminator

. MP-4 3200B

. ( ,

-MC-R 1).

Gel (%[W/V]) (kb)

0.3 5~60

0.6 1~20

0.7 0.8~10

0.9 0.5~7

1.2 0.4~6

1.5 0.2~3

2.0 0.1~2

1 Agarose gel ( 1 )

PCR

>>41

E. marker

marker

. PCR

marker pHY

marker(TaKaRa Code 3404) . 100 bp

DNA Ladder(TaKaRa Code 3407)200bp DNA

Ladder(TaKaRa Code 3410), 500bp simple DNA

Ladder(TaKaRa Code TK3411), 1kb Simple DNA

Ladder(TaKaRa Code TK3422A), 1kb Plus DNA

Ladder(TaKaRa Code TK3412), wide-range DNA

Ladder(TaKaRa Code TK3416b).

2. PCR Agarose agarose

polyacrylamide gel DNA

agarose gel

. agarose 2

.(1) 500 bp Agarose

500 bp

NuSieve 3:1 Agarose(Cambrex) .

agarose 10~1,000 bp

. Southern blotting

. DNA fragment

agarose NuSieve GTG

. NuSieve 3:1 Agarose

1.

(2) Microsatellite DNA Agarose

Microsatellite 4~8%

polyacrylamide gel

MetaPhor Agarose(Cambrex 2~4% gel )

. Agarose 200~800 bp 2%

.

(3) LA-PCR Agarose

LA-PCR 20 kb DNA

pulse field SeaKem Gold Agarose(Cambrex)

. gel gel

Agarose %(kb)

[W/V 1TAE]

NuSieve 3:1

3.0 500~1,000

4.0 100~500

6.0 10~100

NuSieve GTG

2.5 500~1,000

3.5 100~450

4.5 10~100

MetaPhor

2.0 150~800

3.0 100~600

4.0 50~250

5.0 20~130

SeaKem Gold

0.3 5,000~50,000

0.5 1,000~20,000

1.0 400~8,000

2 PCR Agarose

1 Agarose Gel

lane M: pHY marker. lane 1: 600 bp lane 2: 280 bp lane 3: 270 bp (4% NuSieve 3:1 Agarose )lane 1~3 plasmid 600 bp, 280 bp 270 bp DNA.

M 1 2 3

926658489

267

80

600

280270

PCR

DNA

agarose

Polyacrylamide gel

.

STR(short tandem repeat)

.

LA-PCR

PCR

>>42

. 0.4% gel 20 kb

.

Agarose gel agarose

PCR

.

.

PCR

positive control positive

control.

hybridization

.

southern hybridization dot hybridization

.

. Polyacrylamide Gel

Polyacrylamide gel DNA

. gel ,

agarose

. polyacrylamide gel

DNA sequencing, SSCP

DGGE polyacrylamide gel

.

1. Polyacrylamide Gel

(1) Polyacrylamide Gel

Polyacrylamide gel

0.2% (500 bp 1 bp)

, 10 DNA 1 cm well

,

gel DNA agarose

DNA.

(2)

Polyacrylamide gel slab

, mini gel(98 cm).

(3) Polyacrylamide Gel 2)

A.

30% acrylamide(29:1 acrylamide:bis-acrylamide);

acrylamide 29 g, N, N-methylene bis-acrylamide 1

g 100 ml .

Gel (%[W/V]) (bp)

3.5 1,000~2,000

5 80~500

8 60~400

12 40~200

15 25~150

20 6~100

1 Polyacrylamide gel ( 1 )

5TBE

54 g Tris, 27.5 g Borate, 20 ml 0.5 M EDTA(pH

8.0), 1 .

1TBE

5TBE 5 [: 89 mM Tris-

borate, 2 mM EDTA(pH 8.0)].

6 loading buffer

0.25% bromophenol blue, 0.25% Xylene cyanol FF,

30% glycerol.

B. Gel

Glass plate spacer(1 mm )

set.

1gel , 30% acrylamide5

TBE gel

. TBE 1 .

100 ml gel 0.7 ml 10% ammonium

persulfate TEMED(N, N, N,

N-tetra methyl ethylene diamine) 100 ml 35

.

Gel glass plate comb set.

1gel.

C.

Gel plate set 1

TBE, combwellpipette.

30prerunning.

6 loading bufferwell.

PCR

>>43

1~8 V/cm gel.

D.

agarose gel 0.5 /

ethidium bromide . 1 5.5%

polyacrylamide gel VNTR DIS80

.

2. Polyacrylamide Gel

polyacrylamide gel .

(1) DNA Sequencing AP-PCR3) Gel

6~8% acrylamide(19:1 acrylamide:bis-acrylamide),

1TBE, 7 M urea, 20 cm40 cm0.3 mm

gel .

(2) PCR-SSCP4) Gel

5% acrylamide(49:1 acrylamide:bis-acrylamide),

0.5TBE, 5% glycerol, 20 cm40 cm0.3 mm

gel . 4 25.

(3) DGGE5) Gel

6.5~12% acrylamide(37.5:1 acrylamide:bis-

acrylamide), 40~80% (80% : 5.6 M

urea, 32% formamide), 1TAE, 60.

Polyacrylamide gel

agarose

microsatellite

AP-PCR PCR-SSCP

.

. Dot hybridization

PCR DNA

, hybridization

. Dot hybridization

.

hybridization

.

probe southern

hybridization dot hybridization

DNA .

hybridization

. dot

hybridization dot hybridization 1

reverse dot blot

.

1. Dot Hybridization oligonucleotide probe dot

hybridization .

(1) Oligonucleotide Probe

DNA 18~30 probe .

Probe

. 5 32P biotin probe

.

1 Polyacrylamide gel DIS80

lane 1: pBR322/Msp I digest Marker, lane 2: 123 bp ladder Marker, lane3: DIS80 . 5.5% polyacrylamide gel . lane 3 16 band.

1 2 3

622

242

123

67

lane

PCR

>>44

(2) Dot HybridizationA.

96 10

50.1~0.4 N NaOH

100 mM Tris(pH 8.0) acetate

.

Nylon membrane(Amersham Hybond-N+ )

spot.

. Dot

1 cm.

1~2 spot.

20~50 spot

dot blottor . Dot blottor slot-

blot manifold(Schleicher & Schuell) Bio-Dot(Bio

rad).

Nylon membrane 254 nmUV transilluminator

5 DNA . DNA

.

B. Oligonucleotide probe

RI probe 20 pmol probe

T4 polynucleotide kinase, [-32P]ATP.

C. Hybridization

Nylon membrane polyethylene bag ,

prehybridization buffer 42 2

.

[Prehybridization buffer]

5Denhardt's, 5SSC, 0.1% SDS, 0.1 mg/ml

denatured salmon sperm DNA

probe 42 2

, hybridization . Hybridization

oligonucleotide probe Tm ,

hybridization

hybridization.

D.

2SSC, 0.1% SDS102.

0.2SSC, 0.1% SDS 55 15 2

. oligonucleotide probe Tm

hybridization

.

E. AutoradiographyNylon membrane X autoradio-

graphy.

F. RI probe , biotin-labeled probe

hybridization peroxidase-labeled avidin

alkaline phosphatase-labeled avidin ,

(3) Dot hybridization

1 32P probe HPV16 DNA

dot hybridization . HPV16

1, 3, 4 DNA

dot hybridization

HPV16

.

1 Dot hybridization HPV16

((AA)) HPV 16 primer PCR agarose .((BB)) 32P HPV16 oligonucleotide probe dothybridization . lane M: 174 Hae III digestMarker, lane 1: SiHa (HPV16 ), lane 2: HeLa (HPV18 ),lane 3, 4: (HPV16 ), lane 5, 6: condyloma (HPV6/11 ), lane 7, 8: (HPV ). HPV 16 lane 1, 3, 4 140 bp DNA , dot hybridization dot .

(A)M 1 2 3 4 5 6 7 8

(B)1 2 3 4 5 6 7 8

140 bp

PCR

>>45

2. 1Dot HybridizationDot hybridization 1

. Hybridization tetrazolium

probe

hybrid 1 mismatch

hybridization. ras oncogene6)

1 HLA DNA

typing .

oligonucleotide probe 18~20

probe

. probe ASO(allele specific

oligonucleotide) SSO(sequence specific

oligonucleotide) .

3. Reverse Dot BlotHLA typing SSO probe dot

hybridization .

Saiki SSO probe

hybridization reverse dot blot

8).

. SSO probe

SSO probe terminal deoxyribonucleotidyl

transferase T UV

nylon membrane biotin

hybridization . Hybridization

.

Dot hybridization southern hybridization

.

.

tetramethyl ammonium chloride dot

hybridization 1, 1

,

.

1) Sambrook, J., Fritsch, E. F., Maoniatis, T. : in

Moleular Cloning, 2nd edition, pp. 6. 3-6. 35,

Cold Spring Harbor Laboratory Press, NY

(1989)

2) Sambrook, J., Fritsch, E. F., Miniatis, T. : in

Molecular Cloning, 2nd edition, pp. 6. 36-6. 48,

Cold Spring Harbor Laboratory Press, NY

(1989)

3) Welsh, J., McClelland, M. : Nucl. Acids Res.,

1188, 7213-7218 (1990)

4) Hayashi, K. : , 3355, 3085-3090

(1990)

5) Myers, R. M., Maniatis, T., Lerman, L.S.:

Meth. Enzymol., 115555, 501-527 (1986)6) Vries, M. V., Bogaard, M. E., Elst, H., boom, J.

H., Eb, A. J., Bos, J. L. : Gene, 5500, 313-320(1986)

7) Kimura H., et al. : , 3355, 3091-

3103 (1990)

8) Saiki, R, K., Walsh, P. S., Levenson, C. H.,

Erlich, H. A. : Proc. Natl. Acad. Sci. USA, 8866,

6230-6234 (1989)

PCR

>>46

PCR

>>47

1. cycle heat block DNA

PCR.

(1)

94, 30~1 ,

30 .

, microcapillary 1

. 93

, 94 .

GC globin , 93

, 94

. 95 .

globin 1

,

. globin A 93, 1

, B

.

.

, PCR

94, 1 ,

. genome DNA

DNA

5, cycle

.

(2) Annealing

Annealing primer Tm .

1 .

, 2 .

2.

(3)

72 .

, 1 kb 1 ,

1 kb 1 .

Taq polymerase kb

, 3~5 ,

long PCR 10 .

PCR 1.

(4) Cycle

, cycle

, cycle

. PCR

plateau() cycle

. cycle 30

. 30 cycle

35 cycle .

(1)

annealing (1)

(1)

()

94

72

50~60

4

1 cycle 2 cycle cycle

1

PCR

>>48

(5) Cycle

PCR

1 . DNA

, annealing,

,

( 0 1)

. cycle

program.

, 4

.

,

, cycle

.

2.

(1) Primer

Primer 0.5 M . primer

primer

, 0.5 M

*1. 0.5 M ,

0.2 M .

1 mouse genome DNA PCR primer 1 M , 0.5 M .

(2) dNTP

Taq polymerase 0.2 mM dNTP

buffer

. . dNTP

2

.

(3)

100 2.5 U .

2.5 U

. PCR DNA polymerase

5 U/, 2.5 U

micropipette crystal tip

0.5 .

glycerol 0.5

. 1

100

. .

master mix

1

. tube 1

negative control .

(4)

DNA .

, PCR

"SN " . DNA

100 0.1 .

genome DNA (

mol ) , 0.1

single copy .

DNA buffer

PCR . TE DNA

PCR 1/10 PCR

*1.

DNA

DNA

DNA Mg2+ dNTP

. RT-PCR

DNA 50 PCR 1

.

1 TE 10 mM Tris, 1 mM EDTA, PCR 1/10 PCR Tris 10 mM 11 mM, Mg2+ 1.5 mM 1.4 mM , PCR .

(5)

heat block DNA 0.5

microtube 100

. heat block

.

PCR master

mix

. 7

PCR.

PCR

>>49

3. Agarose Gel ElectrophoresisPCR agarose gel electrophoresis

EtBr . PCR

.

PCR ,

. background

10 .

DNA agarose BPB

XC .

gel loading buffer

.

PCR DNA ,

EtBr gel DNA

EtBr

. EtBr gel

DNA ,

. buffer

EtBr , gel EtBr

DNA

. EtBr 4% agarose gel(buffer

: 1TBE) 110 bp DNA 10 bp

2.

gel loading tip mineral oil

.

.

4. Positive ControlNegative Control PCR positive

control negative control

. positive control

.

positive control ,

*1.

PCR DNA ,

DNA

DNA

negative control . PCR

positive control *2.

1 .

2 . . primer , primer PCR . positive control primer .

(1) Negative Control

Negative control

DNA( DNA)

. Transgenic animal

PCR

genome DNA

negative control . RT-PCR

RNA genome DNA

negative control

RNA PCR.

(bp)

396 344 298

220 201

154 134

75

120 bp 110 bp

M 1 2

2

Degenerate PCR 110 bp() cloning. 1 2 110 bp 120 bp . , 110 bp .

PCR

>>50

(2) Positive Control

PCR DNA

.

positive control.

positive control.

positive control

virus PCR

DNA /

positive control. Negative

. positive control

PCR primer

. DNA

positive control

thermal cycler

. DNA

,

positive control .

positive control

(1) , primer, PCR

,

. DNA

. ,

( )

primer

positive control . positive

control , PCR

DNA .

RT-PCR

1st strand cDNA -actin

positive control

.

.

Primer Positive Control

primer RT-PCR

?

, .

. primer

,

primer PCR

.

positive control

primerDNA .

cDNA . primer ? primer positive control ? . positive control . primerPCR positive control . RNA ? RNase RNA .

(3)

Control control

.

negative

control positive control

negative control ,

positive control

.

, positive control

.

1 2 3 negative positive

PCR

>>51

5. DNA - genotyping-

DNA

PCR

(DNA )?

PCR DNA

,

.

genome DNA PCR single copy

. Genome DNA

Proteinase K

.

mouse .

nude mouse

Y

Sry PCR .

XX recipient

XY donor , DNA

haploid genome 0.5 copy .

, haploid 1 copy

. mouse

,

, .

(1)

DNA buffer

150 mM NaCl

10 mM Tris-HCl(pH8.0)

10 mM EDTA

0.1% SDS

Proteinase K stock

20 mg/ml stock

-20

. stock 100

.

PCI

Phenol : Chloroform : Isoamyl alcohol

= 25 : 24 : 1

3 M sodium acetate

100% ethanol

70% ethanol

TE buffer

(2)

(+1) 100 Proteinase K

.

DNA buffer ml

Proteinase K stock 200 /ml

1.5 ml microtube Proteinase K 100

.

( 5~10 )

2) proteinase K *1.

1 heme(hemoglobin )PCR protocol , , heme .

37 2.

( : ~ : )

PCI 100 .

Vortex tube*2.

2 phenol/chloroform , heme .

15,000 rpm, 5.

( : ~ : )

, (

), *3 ,

microtube .

3 heme .

PCR

>>52

~*4.

4 heme phenol/chloroform 2, .

10 3 M sodium acetate .

250 100% ethanol .

10.

( : ~ : )

Microtube 4 15,000 rpm, 15

pellet .

( : ~ : )

micropipette aspirator .

500 70% ethanol vortex

.

Microtube 4 15,000rpm, 5

pellet .

( : ~ : )

micropipette aspirator *5.

5 70% ethanol DNA pellet

Microtube paper towel

.

.

10 TE buffer DNA *6.

6 PCR 1 .

5. PCR

.

.

(1)

Primer

DNA

10 PCR buffer

MgCl2 (25 mM stock solution)

dNTP Mix (2.5 mM stock solution)

Taq polymerase (5 U/)

Mineral oil

(2)

PCR tube *1.

H2O

10 PCR buffer 10

MgCl2 (25 mM stock) 6 (1.5 mM)

dNTP Mix (2.5 mM stock) 8 (0.2 mM)

Primer-1( M stock) (0.5 M)

Primer-2( M stock) (0.5 M)

DNA 1

Taq polymerase (5 U/)*2 0.5 (2.5 U/100 )

total 100

1 1 100 , .

2 Hot start .

50~100 mineral oil *3.

3 .

2~5 ,

PCR *4. Cycle

25~30.

4 annealing cycle , 2 program . program 25~30 cycle .

:

: annealing : cycle :

: annealing : cycle :

:

:

PCR

>>53

PCR 5~10 agarose gel

.

, PCR 1 *5,

15~30 cycle 2nd PCR. 5 1/50~1/100 .

7. Troubleshooting

(1)

primer dimer . (2)

band. (2)

positive control .

) stock ?

) Mg2+ ? 10 buffer Mg2+

) dNTP? Stock

) heat block ? Mineral oil glycerol heat

block tube .

v) Taq polymerase ?

positive control .

) (1)-? (1)-

) DNA? DNA

) RT-PCR, .

Primer positive control .

) (1)-? (1)-

) PCR .

) Primer ? Primer O.D. mol

.

) Annealing ? Tm.

v) ? 94 1.

) Annealing .

) .

) Mg2+ .

) Primer . .

(2) .

Primer ? (0.5 M) (2)

Primer . (0.2 M )

DNA? DNA.

PCR

>>54

? (2.5 U / 100 )

Annealing .

.

Cycle .

Mg2+ .

Hot start.

Primer . .

Nested PCR.

(3) .

stock ?

heat block ? Mineral oil glycerol heat

block tube .

? (4)

(4) Negative control .

RT-PCR negative control . RNase free DNase

. DNA .

DNA.

) Aerosol filter tip .

) Stock DNA ( DNA).

) Polyglove .

) Hood . UV 15 hood

DNA

.

v) Nondisposable . 0.1 N

DNA

. DNA

.

) PCR (). PCR

mol

Micropipette

.

TaKaRa TaqTM TaKaRa Ex TaqTM

PPyyrroobbeessttTTMM DDNNAA PPoollyymmeerraassee

>56

PCR Subcloning

>>57

. PCR Subcloning

1. PCR CloningPCR plasmid subcloning

.

[ 1 : 5.]

DNA oligonucleotide 5

oligonucleotide primer

PCR 5

ligation ( 1).

ligation DNA 5

DNA vector

vector self ligation

vector ligation .

PCR ligation

5( 2).

[ 2 : 3.]

Taq polymerase terminal deoxynucleotidyl

transferase(TdT) .

DNA DNA

blunt end 1 nucleotide

DNA extendase terminal extendase

. Taq polymerase

PCR 3 blunt

endcloning (3).

1

oligonucleotide primer PCR 5.

HOOH

HO

HO

OH

OH

HO OH

OH

HO

P P

HOOH

PP

Vector self ligation . Ligation . Vector self ligation .

HO

HO

OH

OH

PCR

3

53

5

3

PCR Taq polymerase TdT 3 1.

A3

5

5

3A

2

PCR Subcloning

>>58

2. Subcloning PCR cloning

.

TA

cloning

.

(1) Blunt End Ligation

PCR cloning

1. PCR cloning

.

5

PCR blunt end vector

PCR 5. DNA

5 polynucleotide kinase

single strand double strand 5

double strand double strand 3

. PCR 1 3

.

subcloning PCR

primer 5 . Primer

polynucleotide kinase

oligonucleotide amidite

oligonucleotide

.

. 1 3

PCR blunt end vector

.

T4 DNA polymerase 3 5exonuclease

dNTP . Taq polymerase

Klenow DNA

polymerase TdT (

Klenow

). T4 DNA polymerase Pfu polymerase TdT

.

(2)

5

adaptor primer . PCR primer

5 mismatch

primer PCR . PCR

5 .

PCR vector

. 2 primer

vector PCR

.

.

Taq polymerase TdT (STRATEGIES in molecular biology, Vol. 7, p. 8) Taq

polymerase TdT nucleotide

.

.

C G

.

TTA.

TA cloning vector

cloning.

Primer5A.

A

5- A AA - 3

3- T - 5

C

5- C AA - 3 5- C CC - 3

3- G - 5>

3- G - 5

G

5- G GG - 3 5- G AA - 3 5- G CC -3

3- C - 5>

3- C - 5 3- C -5

T

5- T - 3 5- AA - 3

3- A - 5 3- A - 5

PCR Subcloning

>>59

nucleotide

linear strand DNA

base .

BamH I , 5 A

2 oligonucleotide 2

90% 5B1

20 25%

.

A) 5- CGGGATCCCG - 33- GCCCTAGGGC - 5

B) 5- GGGATCCC - 33- CCCTAGGG - 5

CGC

GGATCC

5

3

3

35

53

PCR5

CTTAAGGC

GAATTCCGCTTAAGGC

CGCGGATCCGCGCCTAGG

35

53

GCTTAAp

pAATTCCGGGC

pGATCCG

GAATTC

CCTAGG

CGCGGCGCCTAGp

Vector

5 CGCGGATCC 3

adaptor

(BamH I)

5 CGGGATCC 3

adaptor

(EcoR I)

(BamH I EcoR I)

BamH I EcoR I

2 vector .

4

5

*1 BamH l date . .

Oligonucleotide linear strand

DNA

oligonucleotide

DNA.

1 oligonucleotide

.

Tm

PCR 2 cycle

adaptor primer annealing

Tm adaptor

. 3 cycle

adaptor

annealing

.

(3) Uracil DNA GlycosylaseCloning

2.(2)

adaptor

adaptor dUMP

.

primer PCR primer

5 uracil DNA .

DNA uracil DNA glycosylase(UDG)*1

DNA uracil .

(AP site)

3 12.

BRL UDG cloning vector

3

*2, PCR .

37 ligation

competent

. Insert

vector self ligation

subclone insert .

adaptor

primer vector

.

*1 uracil DNA glycosylase AP endonuclease cytosin amino uracil .*2 3 vector CATCATCATCAT CTACTACTACTA 2 vector PCR .

(4) TA Cloning

1. PCR cloning

Taq polymerase Tth polymerase 1

TdT 4 nucleotide

A

. 3 A 1

PCR Subcloning

>>60

.

oligonucleotide (%) 2 20

Afl III CCACATGTGG > 90 > 90CCCACATGTGGG > 90 > 90

Asc I GGCGCGCC > 90 > 90AGGCGCGCCT > 90 > 90

Ava I CCCCGGGG 50 > 90CCCCCGGGGG > 90 > 90

BamH I CGGATCCG 10 25CGGGATCCCG > 90 > 90

BssH II TTGGCGCGCCAA 50 > 90

Cla I CCATCGATGG > 90 > 90

EcoR I GGAATTCC > 90 > 90CGGAATTCCG > 90 > 90

CCGGAATTCCGG > 90 > 90

Kpn I GGGGTACCCC > 90 > 90CGGGGTACCCCG > 90 > 90

Sac II TCCCCGCGGGGA 50 > 90

Sma I TCCCCCGGGGGA > 90 > 90

Spe I GACTAGTC 10 > 90CGACTAGTCG 10 > 90

Stu I AAGGCCTT > 90 > 90GAAGGCCTTC > 90 > 90AAAAGGCCTTTT > 90 > 90

Xba I GCTCTAGAGC > 90 > 90

Xma I CCCCCCGGGGGG 50 > 90TCCCCCGGGGGGTA > 90 > 90

Uracil adaptor primer

5CAUCAUCAUCAU 3

1

PCR Subcloning

>>61

. 3 T 1

vector PCR vector AT

.

cloning TA cloning TA cloning

vector (FOREX-T vector

TaKaRa code BL001/BL002). vector

PCR subcloning

vector .

vector vector(T vector)

1991 (Marchuk et al. Nucl Acid Res

Vol.1199, p. 1154, 1991)*1, Red Book

Current Protocols in Molecular Biology

. PCR

subcloning .

PPP

OH

CG

PA

PT

PC

PA

PT

PC

PA

PT

PC

PA T

OH

PPPC

G

PA

PT

PC

PA

PT

PC

PA

PT

PC

PA T

P

HO

C

PG

PA

PT

P

PA

PC

PG

PA

PT

P

PA

PC

PG

PA

PT

P

PA

PC

PG

PA

PT

P

PA

P

HO

P

HO

C

PG

PA

PT

P

PA

PC

PG

PA

PT

P

PA

PC

PG

P

A

PT

P

PA

PC

PG

PA

PT

P

PA

P

HO

PG

PT

PA

PG

PT

PA

PG

PT

PA

PG

PT

PA

P

P

HO

C

PG

PA

PT

PU

PA

PC

PG

PA

PT

PU

PA

PC

PG

PA

PT

PU

PA

PC

PG

PA

PT

PU

PA

P

HO

PCR

AP site .

UDG cloning vector

DNA

UDG (37)

AP site (37)

3730

6

PCR Subcloning

>>62

Taq polymerase 4 nucleotide

A nucleotide

dTTP

T 1 . blunt

end vector Taq polymerase dTTP

T vector .

vector 3 self ligation

, 5

PCR cloning . PCR A

5 PCR

ligation concatamer

. vector

PCR

subcloning .

. TA Cloning

1. T Vector

(1)

Plasmid vector

PCI

Phenol : Chloroform : Isoamylalcohol = 25 : 24 : 1

Taq polymerase(5 U/ )

10 Taq buffer

dTTP(100 mM stock )

MgCl2(25 mM stock )

(2)

vector multicloning site blunt end

PT

PT

HO

PP

P

P

P P

OHPP

P

P

P P

OH

HO

PP

P

P

P P

PA

HO

P

P

P

P P

HO

PA

P

P

P

P P

OHPP

P

P

P P

PT

PP

P

P

P P

PPP

T

vector insert

vector

Taq polymerase+dTTP

TA vector

PCR fragment(insert)

ligation

7

PCR Subcloning

>>63

*1

*2.

Vector linear strand III gel

*3.

0.5 microtube*4

.

10 Taq buffer ____ 1

MgCl2(25 mM stock) ____ 1.5 mM

Vector( ____ ) ____ 50 ng/

dTTP(100 mM stock) ____ 2 mM

Taq polymerase(5 U/) ____ 5 U/100

ddH2O ____

total ____

mineral oil .*5

70 2 incubation.

( ___ : ___ ~ ___ : ___ )

Mineral oil 1.5

microtube .*6

PCI vortex .

15,000 rpm 5.

( ___ : ___ ~ ___ : ___ )

()micro tube .

Step ~.

(isopropyl alcohol )*7

3 M Sodium acetate 0.1 vol ( ____ )

Isopropyl alcohol 1 vol ( ____ )

Vortex Mixer 10.

( ___ : ___ ~ ___ : ___ )

15,000 rpm, 4 10

.

( ___ : ___ ~ ___ : ___ )

micropipetting aspiration

.

70% 0.5 ~1 vortex

.

15,000 rpm, 4 2

.

( ___ : ___ ~ ___ : ___ )

micropipetting aspiration

*8.

Microtube paper towel

.

pellet .

50 ng/TE buffer.

(21) 10~*9, -20.

*1 vector .pBluescript , EcoR V Hinc II .

*2 10 .*3 step

.*4 incubator . Thermal Cycler PCR

0.2 microtube . PCR tube .

*5 PCR mineral oil 1 . Mineral oil oil polypropylene tube ( 8).

*6 tube phenol chloroform , mineral oil tube . mineral oil step .

*7 dTTP ethanol isopropylalcohol dTTP .

*8 70% ethanol rinse DNA pellet .

*9 T vector (4. TA cloning (2) ).

2. T Vector Insert

(1)

Mineral oil PCR 5

mini gel

*1.

PCR , 20~50 *2 agarose gel*3

tube

mineral oil

8

band*4.

GelDNAethanol*5.

10 TE buffer ddH2O*6 DNA

, 1~7 *7 ligation.

*1 step .*2 10 cloning .*3 GTG grade agarose .*4 PCR Phenol/Chloroform , ethanol

primer dimer cloning gel . main band band EtBr .

*5 PCR bp low meltingagarose(NuSieve GTG agarose ) phenol PCI .

*6 ligation ddH2O . TE buffer ligation ( ).

*7 ~ PCR .

3. Ligation

(1)

Microtube , ligation

.

10 Ligation Buffer*1 1

ddH2O _____

Vector 1

PCR 1~2

T4 DNA ligase 1

total 10

16 30~1*2 .

Competent cell transformation.

*1 ATP ligation buffer . Buffer ATP ATP .

*2 Ligase maker competent cell condition .

4. TA Cloning (1)T Vector ligation

vector vector self ligation

2 control transformation

vector. colony

T

vector ethanol 1. T Vector

T .

(2)T vector ,

insert self ligation

. T

.

vector . T vector

linear strand DNA

T

vector.

(3)TA cloning insert

random subclone

sequence PCR

.

screening .

(4)PCR vector

2.(2)

primer

adaptor insert

screening .

5. SubcloningPCR subcloning

blunt end ligation

.

blunt end ligation .

T vector blunt ligation

blunt end ligation . Blunt

end DNA T4 DNA polymerase

DNA 1. T Vector

PCR Subcloning

>>64

5 l

10 l20~50 l

stock9~3 l

10 l20~50 l

5 l

ligation1~7 l

PCR Subcloning

>>65

incubation T 3 dTTP

dATP incubation A

ligation .

5 T4 DNA polymerase

Taq polymerase fill-in

A 3 .

5 blunt end

.

Taq polymerase TdT

DNA TA

CG ligation . 9

.

GGATCCCCTAGG

53

53

GATATCCTATAG

53

53

GGATCCCCTAGG

53

53

BamH I

GATATCCTATAG53

53

GGATCACCTAGG

53

53

GGATCAATCCCTAGTTAG

53

53

GATATCCTTTAG53

53

dNTP+Taq Polymerase fill-in

A 3 T 3Ligation

EcoR V

dNTP+Taq polymerase

GCGGCCGCCGCCGGCC

53

53

CAGCTGGTCGAC

53

53

GCGGCCGCCGCCGGCC

53

53

Not I

GATCTGCTAGAC53

53

GCGGCCCCCGCCGGCC

53

53

GCGGCCCCTGCGCCGGGGAC

53

53

GATCTGCTGGAC53

53

dCTP+dGTP+Taq polymerase fill-in

C 3 G 3Ligation

Pvu II

dGTP+Taq polymerase

9

PCR 2 DNA

primer DNA polymerase

primer DNA .

PCRDNA, RNA.

.

PCRDNA,RNA

>>66

PCRDNA, RNA

>>67

(polymerase chain reaction; PCR)

1986. Cold Spring Harbor

Symposium on Quantitative

Biology The Molecular Biology of Homo Sapiens

Kary Mullis PCR 1).

, DNA

cloning DNA target

.

1953 Watson Crick DNA

DNA 20

DNA cloning,

, 10 PCR

. PCR

Mullis 1993 ,

.

DNA

primer DNA hybridize

4 deoxy nucleotide triphosphate(dNTP)

DNA polymerase

primer 3 nucleotide

. PCR

DNA

primer

DNA polymerase DNA

.

DNA . DNA

1976 tRNA

H. G. Khorana MIT

1971 2).

,

PCR.

10, Mullis PCR

. DNA

DNA polymerase

DNA polymerase

.

,

,

.

. PCR

. PCR

PCR DNA, RNA

.

.

Alu PCR, inverse PCR, asymmetric PCR,

multiplex PCR, RT-PCR, PCR ,

Allele PCR, GC clamp PCR, PCR-SSCP

DNA

.

1. PCR DNA polymerase PCR

. PCR

DNA polymerase Thermus aquaticus

Taq DNA polymerase. 2.5 kb

code 832

5 3exonuclease 3 5

exonuclease . AmpliTaq,

TaKaRa Taq clone Taq DNA polymerase

3). DNA

polymerase 1 . Stoffel

fragment Taq 2 PCR

GC rich , 2

. Vent DNA polymerase Thermococcus

litoralis ,

. 3 5exonuclease

primer 3

mismacth DNA .

allele PCR .

Stoffel fragment, Vent DNA polymerase

DNA

DNA sequencing

. Pfu DNA polymerase Pyrococcus

furiosus 3 5exonuclease

DNA

PCRDNA, RNA

>>68

Taq DNA polymerase 12

1 . PCR DNA

. clone

.

, clone clone

. clone

.

2. Long PCR(Long and Accurate PCR) DNA polymerase DNA

5 kb

1~2 kb DNA .

DNA polymerase primer

3 A

mismatch , PCR

DNA purine (depurination), 3

5exonuclease primer 3

DNA

.

PCR 10~20 kb DNA

cDNA genome DNA intron

, DNA

.

1

3 5exonuclease

. Mismatch primer

PCR primer

. 3 5exonuclease

Taq 5 3exonuclease , 3 5

exonuclease Pfu polymerase

4,5).

3. Hot start PCRPCR Tm primer

dimer start . Hot

start .

DNA

polymerase

.

3 . mineral oil

tube ,

DNA (, min) exonuclease mismatch DNA

polymerase (K) () nucleotide/sec 97.5 95.0 92.5 5 33 5 (1 bp/kbp) (kbp)

Taq 94 75~80 150 10 40 130 + - 5~15 5~6

Stoffel 61 50 20 90 ? - - 0.3

Vent ? 80 130 360 ? - + 20 10~13

Pfu 92 72~78 60 180 120 ? ? + 140 1.5~2

rTth 150 + - 5~6

1 DNA polymerase

1 long PCR

(A) Taq DNA polymerase PCR, (B) Taq DNA polymerase 3 5exonuclease polymerase long PCR

primer

primer

primer

DNA

DNA

DNA

3 5exonuclease

PCRDNA, RNA

>>69

tube

. wax beads

.

. wax

.

Taq DNA

polymerase .

polymerase

.

4. Rapid Amplification of cDNA Ends(RACE)cDNA 5

3- PCR6). Anchored

primer7) one-sided PCR8)

.

RACE 2 . cDNA 3

3 35

nucleotide(18 nucleotide Q0, 18 nucleotide Q2

Q0 3 Q1 5

) T 17 QT primer

mRNA

cDNA. cDNAQT primer 5

Q0 primer

mRNA 5

primer-1(GSP1) PCR .

PCR GSP1 GSP2

primer Q1 primer nested PCR

poly(A) 3 cDNA

( 2A). 5 cDNA

mRNA

primer(GST-RT) cDNA

3 terminal deoxynucleotidyl

transferase A . poly(A) GT

primer annealing GSP1 primer PCR

. QT primer GSP2

primer nested PCR cDNA 5

( 2B).

2 RACE 5 DNA

mRNA 5

.

2 RACE cDNA

((AA)) cDNA 3 ((BB)) cDNA 5 QT primer 18 nucleotide Q0, 18 nucleotideQT(Q0 3 Q1 5 nucleotide ) 35 nucleotide Q0-Q1 3 17 T oligonucleotide. 35nucleotide Hind III, Pst I, Xho I . GSP1,GSP2 cDNA primer.GSP1, GSP2 cDNA primer. GST-RT primer

primer

primer

terminal nuleotidyl transferase

primer

PCRDNA, RNA

>>70

3 RACE 9).

cDNA cap full

length mRNA mRNA 5

RNA anchor

. mRNA 5 . full length

mRNA 5 cap

, cap 5

5 .

cap full length

mRNA 5 . 5

RNA T4 RNA ligase

. Anchor mRNA GST-RT

primer cDNA

anchor RNA

NRC1 primerGSP1 primer PCR

. DNA NCR2 GSP2

primer nested PCR full length

mRNA 5 cDNA.

5. Nested PCRNested PCR, seminested PCR 4.

PCR

primer primer

PCR nested PCR,

primer

seminested PCR.

.

6. Arbitrarily Primed-PCR(AP-PCR)AP-PCR primer PCR

5 . cycle (

annealing 50 ) primer

template DNA strand mismatch hybrid

primer .

annealing PCR

DNA . PCR

30 10).

3 RACE

NRC1, 2 mRNA 5 RNA primer. GSP1, 2 primer

T4 RNA ligase

Cap

RNA anchor

1)2) 5cap

terminal nuleotidyl transferase 4 Nested PCR, Seminested PCR

genomeDNA

primer-1 primer-2

primer-3 primer-4

PCRDNA, RNA

>>71

PCR 25 cycles

DNA

.

Polyacrylamide gel finger

print copy

. signal DNA

. AP-PCR

polyacrylamide gel SSCP

DNA

DNA 11).

7. Differential Display RT-PCR(DDRT-PCR),

mRNA 1992

PCR . differential

display RT-PCR(DDRT-PCR)12) RNA arbitrarily

primed PCR (RAP-PCR)13) .

DDRT-PCR 6(A) .

degenerate anchor primer(T)10 MN

cDNA anchor primer

decamer(X)10 primer PCR

cDNA . mRNA poly(A)

annealing oligo dT anchor primer

3 degenerate sequence M N

. M A, G, C , N A, G, T, C

. 12 primer

degenerate primer poly(A)

mRNA cDNA

. cDNA degenerate

arbitrarily primer

genomeDNA

PCR cycle

PCR

6 DDRT-PCR

((AA)) DDRT-PCR . Degenerate anchor primer(T10) T 10 3 2 M, N oligonucleotide M A, G, C , NA, G, T, C . 12 primer .arbitrarily decamer primer(X)10 10 nucleotideoligomer . ((BB)) DDRT-PCR

degenerate anchor primer

arbitrarily decamer primer

A

RNA

downstream degenerateanchor primer

upstream arbitrarilyprimer

PCR, polyacrylamide gel

, PCR 1) northern blot2) cDNA library 3) subcloning

RNA

B

5 AP-PCR

PCRDNA, RNA

>>72

primer downstream primer ,