生物化學(xué)-生化課件chapterDNA重組技術(shù)一靠核酸酶學(xué),二靠堿基配對(duì)語言

Chapter

6ExploringGenesbinant

DNA

technology,

a

newapproach

to

exploring

the

centralmolecules of

lifeA

powerful

means

of yzing

and

altering

genesandproteins.as

a

fruit

of

several

decadesof

basicresearch

onDNA,

RNA

and

es.It

dependson

having

enzymes

that

can

cut,

join,and

replicateDNA

and

reverse-transcribeRNA(restrictionenzymes,

DNA

ligases連接酶,DNApolymerases,reverse

transcriptase,).Thus,

it

is

based

on

nucleic

acid

enzymology.DNA重組技術(shù)一靠核酸酶學(xué),二靠堿基配對(duì)語言A

second

foundationis

thebase-pairing language

that

mediatesnucleic

acid

recognition.hybridization

with

complementary

DNA

orRNA

probes

is

a

sensitive

andpowerfulmeansofdetecting

specific

nucleotide

sequence.與探針雜交In binant

DNA

technology,base

pairing

is

usedto

construct

new

combinations

of

DNA

as

well

as

todetect

and

amplify

particularsequences.This

revolutionary

technologyis

also

criticallydependent

on

the

existence

of

es

andplasmids,三靠載體(

和質(zhì)粒)some

of

the

benefits

of

these

newmethodsthe

discovery

of

restriction

enzymes限制酶led

to

thedevelopment

of

techniques

for

the

rapid

sequencing

ofDNA.A

wealthofinformation

concerning

gene

architecture,thecontrol

of

gene

expression,

andprotein

structure

has

comefrom

thesequencing

DNA

molecules.(限制酶→→各種信息)DNA

molecules

c

so

be

synthesizedde

novo.The

automated

solid-phase

synthesisof

DNA

provideshighly

specific

probes

and

synthetic

tailor-made

genes.

提供高度特異的探針和

量身定做的A

singlecopy

of

a

specific

DNA

sequence

in

acomplexmixture

can

be

amplified

more

than

a

millionfold

by

thepolymerase

chain

reactio

R.the

construction

and

cloning

ofnovel

combinations

of

genesNew

genes

can

be

efficiently

expressed

byhost

cells,

as

exemplified

by

the

production

ofhuman

insulin

bybacteria.Moreover,

specific

mutations

can

be

madein

vitro

to

engineer

proteins

in

designed

ways.

重組→有效表達(dá)→(經(jīng)定向突變)→工程制造特定蛋白探the

Basic

Tools

of

Gene

Exploration索的五大基本工具1.Restriction-enzyme ysis

限制酶分析2.Blotting

techniques

吸印技術(shù)3.DNA

sequencing

DNA4.Solid-phase

synthesis

of

nucleic

acids

核酸固相5.The

polymerasechain

reaction

(PCR)聚合酶鏈反應(yīng)A

final

tool—computer重組DNA技術(shù)的三大基礎(chǔ):限制酶堿基配對(duì)語言載體Restriction

EnzymesSplitDNA

into

Specific

FragmentsRestriction

enzymes,also

called

restrictionendonucleases,限制性核酸酶recognizespecific

base

sequences

in

double-helicalDNA

and

cleave

both

strands

of

the

duplex

atspecific

placesthese

exquisi y

precise

scalpels

(marvelousgifts

of

nature)are

indispensable

foryzing

chromosomestructure,

sequencingvery

long

DNA

molecules.

discovered

by

Werner

Arber,

Hamilton

Smith,and

DanielNathans

in

the

late

1960s.Restriction

enzymes

are

found

in

a

wide

varietyof

prokaryotes.

Their

biologicalroleis

tocleaveforeign

DNA

molecules.The

cell'sown

DNA

is

not

degraded

because

thesitesrecognized

by

its

own

restriction

enzymes

aremethylated甲基化.Many

restriction

enzymes

recognize

specificsequences

of

four

to

eight

base

pairs

and

hydrolyzea

phosphodiesterbond

in

each

strand

in

this

region.A

striking

characteristic

of

these

cleavage

sites

is

thatthey

possesstwofold

rotational

symmetry雙重旋轉(zhuǎn)對(duì)稱.In

otherwords,the

recognized

sequence

ispalindromic回文and

the

cleavage

sites

aresymmetrically

positioned.More

than

90

restriction

enzymes

havebeen

purified

and

characterized.Their

names

consist

of

athree-letter

abbreviation

forthe

host

organism

(e.g.,

Eco

for

Escherichia

coli,

Hinfor

Haemophilus

influenzae噬血桿菌流感

,

HaeforHaemophilusaegyptius埃及噬血桿菌)來源菌菌屬名第一個(gè)字母,種名的前兩個(gè)字母followed

by

astrain

designation菌株株名(if

needed)and

a

roman

numeral

(if

more

than

onerestrictionenzyme

is

produced).羅馬數(shù)字表示來源于同一菌株的兩個(gè)以上的限制酶分離的先后次序The

specificitiesof

several

of

these

enzymes

areshown.Notethatthe

cuts

maybestaggered交錯(cuò)切口or

even平端.Restriction

enzymes

are

used

to

cleave

DNAmolecules

into

specific

fragments

that

are

morereadilyyzed

and

manipulated

than

the

parentmoleculee.g.

the

5.1-kilobase

(kb)

circularduplexDNA

ofthetumor-producing

SV40 is

cleaved

at

1site

byEcoRI,

4

sites

by

HpaI,

and

11sites

by

HindIII.A

piece

of

DNA

producedby

the

action

of

onerestriction

enzymecan

be

specifically

cleavedintosmaller

fragments

by

another

restrictionenzyme.The

pattern

ofsuchfragmentscanserve

asafingerprint

of

a

DNA

moleculeIndeed,

complexchromosomescontaininghundreds

of millions

of

basepairs canbe mappedby

using

a

seriesof

restriction

enzymes.RestrictionFragmentscan

beSeparated

byGelElectrophoresis

andVisualizedSmall

differences

between

related

DNAmolecules

can

be

readily

detected

because

theirrestriction

fragments

can

be

separated

anddisplayed

by

gel

electrophoresisIn

many

types

of

gels,the

electrophoreticmobility

of

aDNA

fragment

is

inversely

proportional

to

thelogarithm

of

the

numberof

base

pairs,up

to

a

certainlimit.DN

段的遷移率與(該片段所包含的)堿基數(shù)的對(duì)數(shù)成反比Polyacrylamide聚丙烯酰胺gels

are

used

toseparate

fragments

containing

up

to

about

1000

basepairswhereas

more

porous

agarose瓊脂糖gels

are

usedto

resolvemixtures

of

largerfragments

(up

to

about20

kb).An

important

feature

of

these

gelsis

their

highresolving

power分辨率.In

certain

kinds

of

gels,fragments

differinginlength

by

just

one

nucleotide

out

ofseveralhundred

can

bedistinguished.Moreover,entire

chromosomes

containing

millionsof

nucleotides

can

now

be

separatedonagarosegels

by

applying

pulsed

electric

fields脈沖電場(chǎng)indifferent

directions.(大片段DNA可因所施加的電流的角度連續(xù)改變而得以分離)Bands

or

spots

ofradioactive

DNA

in

gels

canbevisualized

by

autoradiography放射自顯影.Alternatively,

a

gel

can

be

stained

withethidiumbromide溴化乙啶,

which

fluoresces

an

intenseorange

when

bound

to

double-helical

DNA

雙鏈DNA結(jié)合溴化乙啶后發(fā)出 橙色熒光A

restriction

fragment

containing

aspecificbase sequencecanbeidentified

by

hybridizing it

with

alabeled

complementary

DNA

strandA

mixture

of

restriction

fragments

is

separated

byelectrophoresis

throughan

agarose

gel,

denatured

toform

single-stranded

DNA,

transferred

toaThe

positions

of

thenDiNtrAofcraeglmluelnotssien

tshehegeel

tare

pin

thenitrocellulose

sheetwhere

they

can

be

hybridized

with

a

32P-labeled

single-stranded

DNAprobe.Autoradiography

then

reveals

position

of

the

restriction

fragment

with

asequence

complementary

to

that

of

the

probe.A

particular

fragment

in

the

midst

of

a

million

others

can

readily

beidentified

in

this

way,like

finding

a

needle

in

a

haystack.大海撈針This

powerful

technique

is

known

as

Southern

blotting

(DNA印跡法)because

it

was

devised

by

E.M.

Southern.特定限制片段的鑒定:混合限制片段(雙鏈)經(jīng)agarose分離→變性為單鏈→由膠轉(zhuǎn)移至硝酸纖維素膜→與標(biāo)記的(與特定片段互補(bǔ)的)探針雜交→放射自顯影定位Likewise,

RNA

molecules

can

be

separatedby

gel

electrophoresis,

and

specificsequences

can

be

identified

by

hybridizationThfiosl

onggotursantescfhenriqtouenfiotrrotcheel

syesisofRNA

has

been

whimsically

termed

Northernblotting.A

furtherplay

on

words

accounts

for

the

termWestern

blotting,

which

refers

to

atechniquefor

detecting

a

particular

protein

bystainingwith

specific

antibody.

DNA和RN

段與同位素標(biāo)記的探針雜交后經(jīng)放射自顯影“顯色”,蛋白片段則被特定的抗體“染色”.Southern,

Northern,

andWestern

blots

are also

knownas

DNA,

RNA

and

protein

blots.DNA

can

be

Sequenced

bySpecific

ChemicalCleavage(Maxam-Gilbert

Method)The ysis

of

the

DNA

structure

and

itsrelationship

to

gene

expression

has

alsobeen

markedly

facilitated

by

thedevelopment

of

the

powerful

technique

forThe

chemical

cleavage

methodstarts

with

asingle-sthtraendseedqDuNeAnthcaitnisglaobefleDdNatAonme

eonldewciuthle32sP.Polynucleotide

kinase聚核苷酸激酶is

usually

usedto

add

32P

at

the5’-hydroxyl

terminusThe

labeled

DNA

is

then

broken

preferentially

atoneend

with

the

four

nucleotides.The

conditions

are

chosen

so

thata

age

of

onebrokenis

madeper

chain.(在四種之一的5‘端切斷,控制條件使每條DNA單鏈平均只被切斷一次)In

the

reactionmixture

for

a

givenbase,

theneachbroken

chain

yields

a

radioactive

fragmentextendingfrom

the

32P

label

to

one

of

the

positionsof

the

base,and

such

fragments

are

producedfor

each

positionofthebase.

For

example,if

the

sequence

is5’-

32P

-GCTACGTA-3’the

radioactive

fragments

producedby

specificcleavage

on

the

5’-side

of

each

of

the

fourbaseswouldbeCleavage

at

A:32P

–GCT32P

–GCTACGTCleavageat

G:

32P

–GCTACCleavageat

C:

32P

–G32P

–GCTACleavage

at

T:

32P

–GC32P

–GCTACGThe

fragments

in

ea ixture

are

then

separated

bypolyacrylamide

gel

electrophoresis,

which

can

resolveDNA

molecules

differing

in

length

by

justonenucleotide

.The

next

step

is

to

look

at

an

autoradiogram

of

thisgel.In

this

idealized

example,

the

lowest

band

would

be

inthe

C

lane,

and

the

next

one

up

in

the

T

lane,followedby

one

in

the

A

lane.threenucleotidesentity

of

the

G

at

theHence,

the

sequenceof

thedetected

in

the

5’-CTA-3’

(the5

’

end

is

not

revealed).Reading

all

seven

bands

in

ascending

order

gives

thesequence

5’-CTACGTA-3’.Thus,

the

autoradiogram

of

a

gel

producedfrom

fourdifferent

chemicalcleavages

displays

a

pattern

of

thebands

from

which

the

sequence

can

be

readdirectly.In

practice,

DNA

is

specifically

cleaved

byreagents

that

modify

and

remove

certain

basesfrom

the

sugars先修飾并除去特定堿基,再切斷對(duì)應(yīng)的磷酸二酯鍵Purines

are

damaged

by

dimethylsulfate二甲基硫酸,whichmethylates使甲基化guanine

atN-7

and

adenine

atN-3.The

glycosidic

bond糖苷鍵of

a

methylatedpurine

isreadilybroken

by

heatingat

neutral

pH,which

leaves

the

sugarwithouta

base.二甲基硫酸+加熱(中性pH)→切除嘌呤Subsequentheating

in

alkali

leads

to

the

cleavage

ofthebackbone

at

G

(G),

whereas

treatment

with

dilute

acidcauses

cleavage

at

the

both

A

and

G

(A+G).切除嘌呤后在堿中加熱→G的3’端的磷酸二酯鍵斷開切除嘌呤后在稀酸中加熱→A和G的3’端的磷酸二酯鍵都斷開Cytosine

and

thymine

are

splitoff

byhydrazine肼.The

backbone

is

then

cleavedat

both

C

and

T

bypiperidine哌啶(C+T).肼+哌啶→C和T的3’端的磷酸二酯鍵都斷開Hydrazinolysis肼解inthe

presence

of

2M

NaClspares不切出thymine.Treatment

with

piperidine

then

leads

to

cleavage

onlyat

C

(Ｃ)肼+2MNaCl+哌啶→C的3’端的磷酸二酯鍵斷開Thus,

DNA

can

be

cleaved

at(3’端)only

G,A+G,C

+

T,

and

only

C,

andthe

sequence

can

bededucedby

comparing

the

four

corresponding

lanes

in

anautoradiogram.DNA

is

Usually

Sequenced

byControlled

Termination

ofReplication

(Sanger

DideoxyMethod)受控終止法即Sanger雙脫氧法DNA

c sobe sequenced

bygenerating

through

the

controlledinterruption

ofenzymatic

replication.使酶促

受控中斷In

fact,

this

is

now

the

method

ofchoicebecause

of

its

simplicity.DNA

polymerase I

is

used tocopyaparticular

sequence

of

asingle-stranded DNA

.The

synthesis

is

primed

by

acomplementary

fragment,

whi ay

beobtained

from

a

restriction

enzymedigest

or

synthesized

chemically

.待測(cè)DNA雙鏈經(jīng)變性為單鏈→

(與其側(cè)翼片段互補(bǔ)的)特定引物與之雜交→從引物起在DNA聚合酶指導(dǎo)下,加入dNTP等,并加上一種ddNTP(ddATP,

ddCTP,ddGTP和ddTTP)→每份中DNA的 分別在A,C,G和T的3‘端終止→分四個(gè)泳道走膠→轉(zhuǎn)移,放射自顯影(或熒光顯色)→In

addition

to

the

fourdeoxyribonucleside

triphosphates(radioactivity

labeled),the

incubationmixture

containsa2‘3’-dideoxy og

ofone

of

them

2‘3’-雙脫氧核糖核苷三磷酸The

incorporation

of

thethis ogblocks

furthergrowth

of

the

new

chain

because

it

lacks

the

3‘-hydroxyl

terminus

neededto

form

the

nextphosphodiester

bond.

因其缺少3‘-羥基而無法與下至此中一個(gè)加入的dNATP之間形成磷酸二酯鍵,斷(進(jìn)行到此為止)Hence,

fragments

of

the

various

lengths

areproducedinwhichthe

dideoxy og

isat

the

3'end.Four

such

sets

of

thechain-terminated

fragments(onefor

eachdideoxy og)

are

then

electrophoresed,

andthe

base

sequence

of

the

newDNA

is

read

fromtheautoradiogram

of

the

four

lanes.Fluorescencedetection

is

a

highly

effectivealternative

to

autoradiography.A

fluorescent

tagis

attached

to

anoligonucleotideprimer—a

differently

colored

oneineach

of

thefourchain-terminating

reactionmixtures(e.g.,a

blueemitter

for

termination

at

A

and

a

red

onefortermination

at

C).用四種不同顏色熒光標(biāo)記的引物(primer)代替放射性標(biāo)記Thereaction

mixturearecombined

andelectrophoresed

together.The

separated

bandsof

the

DNA

arethendetected

by

their

fluorescence

astheyemerge

from

the

gel;the

sequence

of

the

their

colorsdirectly

givesthe

basesequence.Sequences

of

the

up

to

500bases

can

bedetermined

inthis

way.Fluorescence

of

the

detection

is

attractivebecausei iminatesthe

use

ofradioactivereagents

and

can

readily

be

automated.Thecompletesequenceof the5386bases

in

φX174

DNA

wasdeterminedThis plishment

is

a

landmark

in

themolecular

biology

because

it

revealed

thetotal

information

content

of

a

DNA

genome.Recently,

a

entire

yeast

chromosome,

310,000base

pairs

long，wassequenced.A

majorgoalnowis

the

sequencingof

the

entirehuman

genome,

which

contains

3.5×109

basepairs.This

task

wascompletedin2002.φX174(5386

bases,1977)(原核)→human

mito.DNA(16569

bp)→Haemophilus

influenzae

桿菌屬流感

(1830137

bp)→bake’s

yeast酵母(真核單細(xì)

胞)(12

millionbp)

→Caenorhabditiselegans線蟲(真核多細(xì)胞)(100

millionbp)

→human(3billion

bp)DNA

Probes

and

Genes

can

beSynthesized

by

AutomatedSolid-phase

MethodDNA

strands,

like

polypeptides

can

besynthesized

by

the

sequentialaddition

of

theactivated

monomers

to

a

growing

chain

that

islinked

to

a

insoluble

support.The

activated

monomers

are

protonateddeoxyribonucleoside

3‘-phosphoramidites亞磷酰胺.

Instep1,

the

3’-phosphorus

atom

of

this ingunites

joined

to

the5‘-oxygen

of

the

growing

chaintoforma

phosphitetriester

1.新加入的(亞磷酰胺脫氧核糖核苷中的)3’-磷原子與(伸長鏈中的)5‘-氧結(jié)合形成亞磷酸三酯.而它的5‘-羥基以及(堿基中的)氨基都被封閉.The

5'-OHof

the

activatedmonomersisunreactivebecause

it

is

blocked

by

a

dimethoxytrityl

(DMT)二甲氧基三苯甲基protecting

group.Likewise,

amino

groups

on

the

purine

and

thepyrimidine

bases

are

blocked.Coupling

is

carried

out

under

anhydrous無水conditions

because

water

reacts

withphosphoramidites.In

step

2,the

phosphitetriester

(in

waterP

istrivalent)is

oxidized

by

iodine碘to

formaphosphotriester(in

whichP

ispentavalent)2.亞磷酸三酯(三價(jià)磷)被碘氧化成磷酸三酯(五價(jià)磷).In

step3,theDMT

protectinggrouponthe

5‘-OH

of

thegrowing

chainis

removed

byaddition

of

the

dichloroacetic

acid二氯乙酸,which

leaves

other

protecting

groupsintact.3.加入二氯乙酸以去掉其5‘-OH

的保護(hù)基DMTThe

DNA

chain

is

now

elongated

by

one

unit

and

readyfor

another

cycle

of

addition.Each

cycle

takes

only

about10

minutesand

elongates

more

than

98%

of

the

chains.This

solid-phase

approach

is

ideal

for

the

synthesis

ofDNA,

as

it

is

for

polypeptides,

becausethe

desiredproduct

stays

on

the

insolublesupport

until

thefinalrelease

step.All

the

reactions

occur

in

a

single

vessel,

and

excess

solublereagents

can

be

added

to

drive

reactions

to

completion.At of

each

step,

soluble

reagents

and

byproducts

areof

the

synthesis，NH3

is

added

to

removed

allpro groups

and

release

the

oligonucleotide

from

the

solidwashed

away

from

the

glass

beads

that

bear

the

growing

chains.Atsupport.Because

elongation

is

never

100%complete,

the

new

DNAchains

are

of

the

diverse

lengths—the

desired

chain

is

thelongest

one.因?yàn)槊看紊扉L都無法100%實(shí)現(xiàn),所以最終產(chǎn)物中只有最長的DNA才是所需的鏈.The

sample

can

be

purified

by

high-performance

liquidchromatography

or

by

electrophoresis

on

polyacrylamide

gels.DNA

chains

up

to

100

nucleotides

long

can

readily

besynthesized

by

this

automated

method.The

ability

to

rapidly

synthesize

DNA

chains

of

the

anyselected

sequence

opens

many

experimental

avenues.e.g.,

an

oligonucleotide

labeled

at

one

end

with

32P

or

afluorescent

tag

can

be

used

to

search

for

a

complementarysequencei y

long

DNA

molecules

or

even

in

agenomeconsisting

o ychromosomes.同位素或熒光標(biāo)記的人工

很大的DNA分子中甚至整個(gè)寡聚核苷酸用來“大海撈針”,從組中“釣”出與之互補(bǔ)的序列The

use

of

the

labeledoligonucleotidesas

DNA

probes

ispowerful

and

general.e.g.,

a

DNA

probethat

isbase-paired

to

aknowncomplementary

sequence

in

a

chromosome

can

serve

asthe

starting

point

of

an

explorationof

adjacent

unchartedDNA.

(“

步查”:用一已鑒定的 作探針釣出含有它鄰近序列的克隆.后者再作為探針鑒定含有與之鄰近序列的克隆.如此進(jìn)行.每輪雜交,便由已鑒定 起沿著 向前“走一步”.)Such

a

probe

can

be

used

as

a

primerto

initiate

thereplicationof

neighboringDNA

by

DNA

polymerase.

(探針側(cè)翼DNA的

)One

of

the

most

exciting

applications

ofthe

solid-phase

approach

is

the

synthesisof

the

new

tailor-made

genes.New

proteins

with

novel

propertiescan

now

be

producedin

abundanceby

expressing

synthetic

genes.

Proteinengineering

has e

areality.DNA

can

be

Sequenced

byHybridizationto

Oligonucleotide

Arrays寡聚核苷酸方陣(DNA

Chips

)The

synthesis

of

the

arrays

ofolignucleotides

makes

it

feasibletosequenceDNA

in

a

new

waySequencing

by

hybridization

(SBH)雜交

uses

aset

of

the

olignucleotide

probes

of

definedsequenceto

search

for

complementary

sequenceson

a

longerDNA

moleculeSuppose

that

a

12-mer-DNA

withthe

sequence5'-AGCCTAGCTGAA-3'is

mixed

with

a

complete

set

of

octanucleotide

probes.Only

5

of

the

65,536(48)

octamers

would

form

hybridsif

perfect

complementarity

were

required.(一條)待測(cè)的十二聚核苷酸與全部(共48條)八聚核苷酸混合,其中只有五條與之完全互補(bǔ)The

sequence

of

the

12-mer

can

then

besequencesreconstructed

by

aligning

theoverlapof

the

hybridizing

oligomers:3'-TCGGATCG-5'CGGATCGAGGATCGACGATCGACTATCGACTT3'-TCGGATCGACTT-5'5'-AGCCTAGCTGAA-3'In

practice,

the

hybridization

patternis

more

complexbecause

oligonucleotides

that

are

not

perfectlycomplementary

to

the will

still

formduplexes,though

with

lower

affinity.Restriction

Enzymes

and

DNA

Ligase“剪刀和漿糊”are

Key

Tools

in

Formingbinant

DNA

Moleculesthe

development

of binant

DNA

technology

hasrevolutionized

bioch y.

New

combinations

of

the

unrelatedgenes

can

be

constructed

in

the

laboratory

by

it.These

novel

combinations

can

be

cloned—amplified

manifold—byintroducing

them

into

suitable

cells,

where

they

are

replicated

by

theDNA

synthesizingmachineryof

the

cellsofthe

host.新的重組DNA被(宿主細(xì)胞的DNA

機(jī)器)克隆放大The

inserted

genes

are

often

transcribed

and

translated

in

their

newsetting.所

在新的駐地轉(zhuǎn)錄翻譯What

is

most

striking

is

that

t etic

endowment

of

the

host

can

bepermanently

altered

in

a

designed

way.

（這種遺傳處理結(jié)果）在宿主中成為

性改變How

can

novel

DNA

molecules

be

constructed

in

thelaboratory?DNA

vector

canreplicate

autonomously自主

in

anappropriatehost.Plasmids

and

λ

phage

(a )are

choicevectorsforcloning

in

E.coli.兩類重要載體:質(zhì)粒和嗜菌體The

vector

can

be

prepared

for

splicing

by

cleaving

it

at

asingle

specific

site

with

a

restriction

enzyme.the

staggered

cuts

are

known

ascohesiveends粘性末端歌

(載體和用同一限制酶切割獲得的DNA互補(bǔ),連接)Any

DNA

fragment

(prepared

by

using

the

same

restrictionenzyme)

can

be

inserted

into

this

plasmid

if

it

has

the

samecohesiveends.The

single-stranded

ends

of

the

fragmentandthecutplasmidcan

be

annealed

and

thenjoined“退火”連接byDNA

ligase,連接酶which

catalyzesthe

formation

of

aphosphodiester

bond

at

a

breakin

a

DNA

chain.DNA

ligase

requires

a

free3‘-OH

group

and

a5’-phosphategroup.連接酶催化雙鏈DNA中磷酸二酯鍵的生成（3‘-OH+5’-磷酸基）the

chains

joined

by

ligase

must

be

in

a

double

helixThis

cohesive-end

method

for

joining

DNA

moleculescan

be

made

general

by

using

a

short

chemicallysynthesized

DNA

linker短接頭

that

can

be

cleavedby

restrictionenzymes.,

the

linker

is

covalently

joined

to s

of

a

DNA

fragment

orvector.For

example,the

5‘-ends

of

a

decameric

linker

and

a

DNA

molecule

arephosphorylated

by

polynucleotide

kinase

多聚核苷酸激酶(使之磷酸化)and

then

joined

by

the

ligase

from

T4

phage.來自T4噬菌體的連接酶.1.

上述兩個(gè)酶使(人工

的)短接頭與DN

段或載體末端(磷酸化并)共價(jià)連接This

ligase

can

form

a

covalent

bond

between

blunt-ended平端

(flush-ended)double-helical

DNA

molecules.Cohesive

ends

are

produced

when

these

terminal

extensions

are

cut

byan

appropriate

restriction

enzyme.2.用合適的限制酶(在短接頭范圍內(nèi))切出粘性末端Thus,

cohesive

ends

corresponding

to

a

particular

restriction

enzyme

can

be

added

to

virtually

any

DNA

molecule.

任何DNA分子的末端都可形成(由特定的限制酶切割的)粘性末端We

see

here

the

fruits

of

combining

enzymatic

and

synthetic

chemicalapproaches

in

crafting

new

DNA

molecules.Plasmids

and

Lamda

Phage

areChoice

Vectors

for

DNA

Cloning

inBacteriaMany

plamids

and

bacteriophageshave

bmodified

to

enhance

the

deliveryof

thengeniouslybinant

DNAmolecules

into

bacteriaand

to

facilitate

t lection

of

thebacterialharboring

them.載體經(jīng)巧妙修飾以：1.增強(qiáng)遞送重組DNA進(jìn)入宿主細(xì)胞的能力2.促進(jìn)宿主細(xì)菌對(duì)載體的選擇Plasmids

are

circular

duplexDNA

molecules

occurringnaturallyin

some

bacteria(2~several

hundredkb).

質(zhì)粒是天然存在在某些細(xì)菌中的閉合雙鏈DNA分子They

carry

genesfor

the

inactivationofantibiotics抗生素失活,the

production

of

thetoxins制造毒素,and

thebreakdownof

the

natural

products降解天然產(chǎn)物.These

accessory輔助

chromosomes

canreplicateindependentlyofthehostchromosome.質(zhì)?？稍谒拗黧w中作為輔助

自主In

contrast

with

the

host

genome,they

are

dispensable不必要under

certain

conditions.Plasmid

pBR322

contains

genes

for

totetracycline四環(huán)素and

ampicillin氨芐青霉素(抗性）.This

plasmid

can

be

cleaved

at

a

variety

of

uniquesites

by

different

endonucleasesInsertion

of

the

DNA

at

the

EcoRI

restriction

site

doesnotalter

either

of

t es

for

antibiotic

.Insertion

at

the

HindIII,

SaII,

or

BamHI

restriction

siteinactivates

t efortetracycline ,an

effectcalled

insertional

inactivation

失活.Cells

containing

pBR322

with

a

DNA

insert

at

one

of

theserestriction

sites

are

resistant

to

ampicillin

bu sitive

totetracycline只抗氨芐青霉素不抗四環(huán)素,and

so

they

can

bereadily

selected.Cells

that

failed

totakeupthevector

are

sensitive

to

bothantibiotics,

whereas

cells

containing

pBR322

without

aDNAinsertare

resistanttoboth.

1.質(zhì)粒轉(zhuǎn)染失敗(未進(jìn)入):細(xì)菌對(duì)兩種抗生素都敏感;

2.質(zhì)粒轉(zhuǎn)染成功(進(jìn)入,但未含

DNA

)細(xì)菌對(duì)兩種抗生素都具抗性;

3.質(zhì)粒轉(zhuǎn)染成功(且在抗四環(huán)素的上述切點(diǎn)

了外源DNA):僅抗青霉素Lambda

(λ)

phaoys

a

choiceof

life

styles:

it

can

destroyits

host

or

it

canepart

of

its

host

(the

lytic裂解pathwayand

the

lysogeni

溶源pathway).Mutant

λ

phages

designed

for

c