變性梯度凝膠電泳CRDGGE

變性梯度凝膠電泳CRDGGE變性梯度凝膠電泳(denatured

gradient

gelelectrophoresis，DGGE)最初是Fisher和Lerman等人于20世紀(jì)80年代初期發(fā)明的，起初主要用來檢測(cè)DNA片段中的點(diǎn)突變。Muyzer等人在1993年首次

將其應(yīng)用于微生物群落結(jié)構(gòu)研究。后來又發(fā)展出其衍生技術(shù)，溫度梯度凝膠電泳（temperature

gradientgel

electrophoresis，TGGE）。此后十年間，該技術(shù)被廣泛用于微生物分子生態(tài)學(xué)研究的各個(gè)領(lǐng)域，目

前已經(jīng)發(fā)展成為研究微生物群落結(jié)構(gòu)的主要分子生

物學(xué)方法之一。1.

Introduction變性梯度凝膠電泳（DGGE）:一種分離相似大小DNA片段

的電泳方法。即雙鏈DNA在變性劑(如尿素和甲酰胺)濃度或溫度梯度增高的凝膠中電泳，隨變性劑濃度升高，由于Tm值不同，DNA的某些區(qū)域解鏈，降低其電泳泳動(dòng)性，導(dǎo)致遷移率下降，從而達(dá)到分離不同片段的目的。由于各類微生物（如細(xì)菌和古細(xì)菌）的16sRNA基因序列中可變區(qū)的堿基順序有很大的差異，其中不同土壤微生物的16sRNA基因的V3區(qū)擴(kuò)增的DNA片斷在DGGE中的應(yīng)用最為廣泛，根據(jù)電泳條帶的多寡和條帶的位置可以初步辨別出樣品中微生物的種類多少，粗略分析土壤樣品中微生物的多樣性。變性梯度凝膠電泳(DGGE)The

methods

of

detecting

single

basemutationsSouthern

blottingSingle-Strand

Conformational

Polymorphism(SSCP,單鏈構(gòu)象多態(tài)性分析)Denaturing

Gradient

Gel

Electrophoresis(DGGE,變性凝膠梯度電泳)Carbodiimide(CDI，碳化二亞胺檢測(cè)法)Chemical

Cleavage

of

Mismatch(CCM，化學(xué)切割錯(cuò)配法)Rnase

cleavage（RNA酶裂解法）Heteroduplex

analysis（異源雙鏈分析）the

ProteinTruncation

Test(PTT，蛋白截短測(cè)試)Temporal

Temperature

Gradient

Gel

Electrophoresis(TTGE,時(shí)間溫度梯度電泳).yearpercent%number20014.75%2120023.85%1720036.79%3020049.05%40200511.09%49200612.90%57200715.84%70200815.16%67200913.12%5820107.47%33in

all100%442①

the

papers

which

titles

contain

‘DGGE’

in

2001-2010The

article

about

DGGEyearPercent%number20014.63%18420024.48%17820036.06%24120047.52%29920059.23%367200610.99%437200713.13%522200815.39%612200918.86%75020109.73%387in

all100%3977②

the

papers

which

themes

contain

‘DGGE’

in

2001-20102008-2010

Microbiological

characterisation

of

Robiola

di

Roccaveranocheese

using

PCR-DGGE.

Bonetta,

S.Carraro,

E.Rantsiou,

K.Cocolin,

L.

2008Food

Microbiology.(IF

2.847)Variation

in

the

active

diazotrophic

community

in

rice

paddy-

nifH

PCR-DGGE

analysis

of

rhizosphere

and

bulk

soil.Wartiainen,

I.

;Eriksson,

T.

;Zheng,

W.

W.

;Rasmussen,

U.2008Applied

Soil

Ecology.(IF2.247)Analysis

of

community

structure

of

a

microbial

consortiumcapable

of

degrading

benzo(a)pyrene

by

DGGE.

Luo,

Y.

R.

;Tian,

Y.

;Huang,

X.

;Yan,

C.

L.

;Hong,

H.

S.

;Lin,

G.

H.

;Zheng,

T.

L.

2009Marine

Pollution

Bulletin(IF

2.63)Bulk

soil

and

rhizosphere

bacterial

community

PCR-DGGE

profiles

and

beta-galactosidase

activity

asindicators

of

biological

quality

in

soils

contaminatedby

heavy

metals

and

cultivated

with

Silene

vulgaris(Moench)

Garcke.

Martinez-Inigo,

M.J.

;

PerezSanz,

A.;

Ortiz,

I.

;Alonso,

J.

;Alarcon,

R.

;Garcia,

P.

;Lobo,

M.C.Chemosphere(IF3.253)

2009

Application

of

real-time

PCR,

DGGE

fingerprinting,and

culture-based

method

to

evaluate

theeffectiveness

of

intrinsic

bioremediation

on

thecontrol

of

petroleum-hydrocarbon

plume.

Kao,

C.

M.

;Chen,

C.

S.

;Tsa,

F.

Y.

;Yang,

K.

H.

;Chien,

C.

C.

;Liang,

S.

H.

;Yang,

C.

A.

;Chen,

S.

C.

2010Journal

of

Hazardous

Materials(IF

4.144)Comparative

analyses

of

amplicon

migration

behavior

indiffering

denaturing

gradient

gel

electrophoresis

(DGGE)systems.

Thornhill,

D.

J.

;Kemp,

D.

W.

;Sampayo,

E.

M.

;Schmidt,

G.

W.

2010Coral

Reefs(IF

3.056)Characterization

and

biotechnological

potential

ofpetroleum-degrading

bacteria

isolated

from

oil-contaminated

soils.

Zhang,

Z.

Z.

;Gai,

L.

X.

;Hou,

Z.

W.

;Yang,

C.

Y.

;Ma,

C.

Q.

;Wang,

Z.

G.

;Sun,

B.

P.

;He,

X.

F.

;Tang,

H.

Z.

;Xu,

P.Bioresource

Technology(IF

4.253)In

a

denaturing

gradient

acrylamide

gel,

double-stranded

DNA

is

subjected

to

an

increasingdenaturant

environment

and

will

melt

in

discretesegments

called

"melting

domains".The

melting

temperature

(Tm)

of

these

domains

is

sequence-specific.

When

the

Tm

of

the

lowest

melting

domain

isreached,

the

DNA

will

become

partially

melted,

creatingbranched

molecules.

Partial

melting

of

the

DNA

reduces

itsmobility

in

a

polyacrylamide

gel.2.The

principle

of

DGGE·Since

the

Tm

of

a

particular

melting

domain

issequence-specific,

the

presence

of

a

mutation

willalter

the

melting

pro

that

DNA

when

compared

towild-type.

DNA

containing

mutations

will

encountermobility

shifts

at

different

positions

in

the

gel

thanwild-type.

If

the

fragment

completely

denatures,

themigration

again

becomes

a

function

of

size(Fig

1)·In

DGGE,

the

denaturing

environment

is

created

bya

combination

of

uniform

temperature,

typicallybetween

50

and

65℃

and

a

linear

denaturantgradient

formed

with

urea

and

formamide.

A

solutionof

100%

chemical

denaturant

consists

of

7

M

ureaand

40%

formamide.Fig.1.

An

example

of

DNA

melting

properties

in

a

perpendicudenaturing

gradient

gel.

At

a

low

concentration

of

denaturanthe

DNA

fragment

remains

double-stranded,

but

as

theconcentration

of

denaturant

increases,

the

DNA

fragment

beginto

melt.

Then,

at

very

high

concentrations

of

denaturant,the

Dfragment

can

completely

melt,

creating

two

single

strands.The

denaturing

gradient

may

be

formedperpendicular

or

parallel

to

the

directionofelectrophoresis.（1）A

perpendicular

gradient

gel,

in

which

thegradient

is

perpendicular

to

the

electric

field,

typicuses

a

broad

denaturing

gradient

range,

such

as

0–100%or

20–70%.（2）In

parallel

DGGE,

the

denaturing

gradient

isparallel

to

the

electric

field,

and

the

range

ofdenaturant

is

narrowed

to

allow

better

separation

offragments.Examples

of

perpendicular

and

parallel

denaturinggradient

gels

with

homoduplex

and

heteroduplexfragments

are

shownin

Figure

2Fig.

2.

A.

Perpendiculardenaturinggradient

gel

B.

ParalleldenaturinggrThe

melting

behaviour

of

DNA

fragments,

aswell

as

the

optimal

gradient

can

be

determinexperimentally

with

perpendicular

gradientPerpendicular

gels

have

an

increasing

gradieof

denaturants

or

temperature

from

left

to

riperpendicular

to

the

direction

of

electrophThe

sample

is

applied

across

the

entire

widtthe

gel

and

electrophoresed

for

about

4

hourscertain

Volts.By

using

DGGE

or

TGGE,

50%

of

the

sequencevariants

can

be

detected

in

DNA

fragments

up

to

50bp.

This

percentage

can

be

increased

to

nearly

10by

the

attachment

of

a

GC

rich

sequence

to

onside

of

the

DNA

fragmentA

sequence

of

guanines

(G)

and

cytosines

(C)

isadded

to

the

5’end

of

one

of

the

PCR

primers,

co-amplified

and

thus

introduced

into

the

amplifiedfragments.

The

GC

rich

sequence

acts

as

a

highmelting

domain

preventing

the

two

DNA

strandsfrom

complete

dissociation

into

single

strandslength

of

the

GC

clamp

can

vary

between

30

and

50nucleotides.GC

clampFig.

3.

An

example

of

wild-typand

mutant

DNA

fragments

thatwere

denatured

and

re-annealeto

generate

four

fragments:

theteroduplexes

and

twohomoduplexes

run

on

a

paralledenaturing

gradient

gel.

Themelting

behavior

of

theheteroduplexes

is

altered

sothey

melt

at

a

lowerdenaturant

concentration

thathe

homoduplexes

and

can

bevisualized

on

a

denaturinggradient

gel.DNA

bands

in

DGGE

and

TGGE

profilescan

be

visualised

using

ethidiumbromide(EB)

or

SYBR

GREENⅠ.A

more

sensitive

detection

method

is

silver

staihowever,

silver

staining

also

stains

singlestrandedDNA,

and

silver

stained

gels

cannot

be

usfor

subsequent

hybridization

analysisDGGE圖譜的分析圖像的采集和條帶的回收DGGE分析環(huán)境樣品基因組DNA的提取目標(biāo)片段的PCR擴(kuò)增電泳前準(zhǔn)備工作電泳分析剝膠、染色DGGE條帶測(cè)序3.DGGE分析微生物群落的主要步驟Flow

diagram

of

the

different

steps

ithe

study

of

the

structure

andfunction

of

microbial

communitiesGenetic

finger-printing

by

DGGE

orTGGE

of

molecular

markers

is

theheart

of

a

strategy

to

study

thepresence

(DNA)

and

activity

(rRNA

or

mRNA)

of

bacterial

populations

incomplex

mixtures.

Additionalinformation

about

particular

bacterpopulations

within

the

community

canbe

obtained

by

hybridisation

analysiwith

taxon-specific

probes.Furthermore,

individual

bands

can

beexcised

from

the

gels

and

sequencedtoidentify

the

community

members.These

techniques

are

also

used

tomonitor

the

success

of

isolation

ofbacteria

in

pure

cultures,

and

toscreen

clone

libraries

for

redundanc總DNA的提取是D

GE研究微生物群落的基礎(chǔ)。適合的DNA提取方法對(duì)環(huán)境樣品微生物群落結(jié)構(gòu)分析非常重要。適合DNA提取方法的考量：①DNA的得率。②能否進(jìn)行PCR擴(kuò)增以及擴(kuò)增的重復(fù)性。③制備DNA花費(fèi)時(shí)間的長(zhǎng)短。關(guān)于DNA提取的建議：優(yōu)先考慮基于原位裂解的方法，根據(jù)實(shí)際情況采用酶解/酚氯仿抽提法，或者DNA提取試劑盒（建議購(gòu)買Qiagen公司相關(guān)產(chǎn)品）

。環(huán)境樣品基因組DNA的提取選擇適合的目標(biāo)片段，設(shè)計(jì)合適的引物，注意有GC夾子情況下引物二聚體的行程。環(huán)境樣品目標(biāo)片段的擴(kuò)增最好采用Touch

downPCR。注意PCR的環(huán)境，V3區(qū)產(chǎn)物擴(kuò)增極易污染。目標(biāo)片段的PCR擴(kuò)增細(xì)節(jié)決定成??！DGGE分析從這一步開始，帶上手套。配置試劑時(shí)一定要用去離子水，可以配置不同梯度的變性溶液備用，注意變性溶液中大顆粒的過濾及脫氣。制膠洗膜時(shí)用的各個(gè)容器要用去離子水洗滌干凈，以防止氯離子污染。灌膠前準(zhǔn)備好所有需要的東西，試劑、槍頭，甚至物品擺放的位置。制膠是實(shí)驗(yàn)的關(guān)鍵，在往玻璃板中灌膠時(shí)要?jiǎng)蛩俚剞D(zhuǎn)動(dòng)滑輪，將凝膠液勻速地灌入玻璃板。灌膠后立刻清洗注射器，以防丙烯酰胺凝固，堵塞管子。DGGE前的準(zhǔn)備工作DGGE制膠主體部件：上樣的膠孔要用去離子水沖洗干凈并吸干。PAGE膠裝入電泳支架時(shí)注意用去離子水潤(rùn)滑橡膠墊。裝入后在膠孔中加入緩沖液。上樣時(shí)上樣器要深入膠孔底部。盡量在同一個(gè)膠上比較所有樣品，每一個(gè)膠上要有

marker

lane。將膠放入電泳槽中時(shí)注意正負(fù)極。建議低電壓電泳一段時(shí)間，在樣品完全進(jìn)入膠中之后再升電壓。電

泳停止電泳后注意把電泳儀調(diào)零之后在關(guān)閉電源剝膠需要細(xì)致，用好去離子水和保鮮膜。染色前做好膠樣品順序的標(biāo)記。銀染、EB染色和SYBRGreen染色。剝膠與染色——垂直電泳凝膠變性梯度的確定關(guān)于PCR產(chǎn)物的純化和定量關(guān)于DGGE

marker的制作獲得高質(zhì)量的DGGE圖譜。

DGGE條帶的回收－－注意紫外條件下操作的安全。－－盡量減少DNA在紫外下的照射時(shí)間。－－不同長(zhǎng)度目標(biāo)片段從切膠條帶中的回收。測(cè)序注意要點(diǎn)－－回收條帶的DNA在PCR擴(kuò)增后，一定要克?。_認(rèn)克隆與母條帶可以跑到同一個(gè)位置后，再送去測(cè)序－－每個(gè)條帶至少測(cè)3個(gè)克隆圖像的采集和條帶的回收DGGE圖譜的聚類分析、相似性分析DGGE圖譜的分析DGGE圖譜的主成分分析DGGE圖譜的數(shù)字化——Quantity

oneDGGE圖譜的數(shù)字化后的主成分分析用Quantity

One（Bio-Rad，USA）軟件對(duì)D

GE圖譜進(jìn)行數(shù)字化處理。按照如下公式計(jì)算多樣性指數(shù)（Sha

non-Wiener

index）其中，s代表每泳道中的條帶數(shù)量；Pi為泳道中第i條帶灰度（height

of

the

peak）占該泳道總灰度的比例。菌群均勻度（eve

ne

s，E）：E=H′/H′max，H′max=ln

S多樣性指數(shù)的計(jì)算4.

DGGE的優(yōu)缺點(diǎn)優(yōu)點(diǎn)(1)DGGE的最大優(yōu)點(diǎn)就是無需進(jìn)行微生物培養(yǎng)，且能檢測(cè)出難以或不能培養(yǎng)的微生物，同時(shí)檢測(cè)多種微生物突變檢出率高。DGGE的突變檢出率為99%以上幾乎可以檢出所有突變可將突變分子完好無損地同野生型分子分開用于進(jìn)一步的分析無須標(biāo)記。DGGE不需同位素?fù)饺?，可避免同位素污染及?duì)人體造成的傷害。（5)操作簡(jiǎn)便、快速、重復(fù)性好、結(jié)果準(zhǔn)確可靠。電泳前只需一步操作，DGGE一般在24小時(shí)內(nèi)即可獲得結(jié)果。PCR-DGGE方法則能客觀完整地鑒定微生物，根據(jù)參考菌株和樣品16S

rRNA的PCR產(chǎn)物在凝膠中的相對(duì)位置來進(jìn)行判斷．若割膠測(cè)序，然后序列比對(duì)，就可以得出遺傳相關(guān)性（6)可用于未經(jīng)擴(kuò)增的基因組DNA,可檢測(cè)出象甲基化這樣的DNA修飾缺點(diǎn)(1)只能分離較小的片段(<500

bp),對(duì)于大片段的分離效率下降(2)DGGE圖譜中單一的條帶并不總是代表單一的菌株,或是在不同的泳道中移動(dòng)到同一位置的條帶可能由不同的細(xì)菌組成

(3)DGGE通常顯示群落中優(yōu)勢(shì)種類的r

DNA片段,只有占整個(gè)群落細(xì)菌數(shù)量約l%或以上的類群能夠通過DGGE檢測(cè)到。由于某些種類的16S

r

DNA不同拷貝之間的多態(tài)性問題,可能導(dǎo)致自然群落中細(xì)菌數(shù)量的過多估計(jì)。DGGE技術(shù)對(duì)微生物的分類鑒定依賴于基因數(shù)據(jù)庫(kù),若數(shù)據(jù)庫(kù)中基因序列信息不夠豐富,將會(huì)限制DGGE的使用。需要專門設(shè)備，用計(jì)算機(jī)對(duì)序列進(jìn)行分析，需要進(jìn)行預(yù)實(shí)驗(yàn)昂貴的“GC夾板”用含有毒性物質(zhì)甲酰胺的梯度凝膠無法確定突變?cè)贒NA片段中位置附：DGGE操作步驟將海綿墊固定在制膠架上，把類似‘三明治’結(jié)構(gòu)的制膠板系統(tǒng)垂直放在海綿上方，用分布在制膠架兩側(cè)的偏心輪固定好制膠板系統(tǒng)，注意一定是短玻璃的一面正對(duì)著自己。共有三根聚乙烯細(xì)管，其中兩根較長(zhǎng)的為15.5cm