Download - Amplified Fragment Length Polymorphism (Aflp)

7/25/2019 Amplified Fragment Length Polymorphism (Aflp)

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AMPLIFIED FRAGMENTLENGTH POLYMORPHISM

(AFLP)

ANNA KHUZAIMAHP200310017


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Amplifed Fragment LengthPolymorphism PCR(or AFLP-PCRor ju!AFLP) " # P$R%'!oo u' "* +'*'!",r''#r,-. DNA /*+'rr"*!"*+. #* "* !-'

r#,!",' o +'*'!", '*+"*''r"*+

D''o' "* !-' '#r4 1550 &4 K'4+'*'61. AFLP u' r'!r",!"o* '*849'!o "+'!

+'*o9", DNA. oo:' &4 "+#!"o*o##!or !o !-' !",;4 '*o !-'r'!r",!"o* r#+9'*!
http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/DNA_fingerprinthttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://c/Documents%20and%20Settings/ANNA.ANNA-257E744327/My%20Documents/Amplified_fragment_length_polymorphism.htmhttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Ligationhttp://en.wikipedia.org/wiki/Sticky_endshttp://en.wikipedia.org/wiki/Restriction_fragmenthttp://en.wikipedia.org/wiki/Restriction_fragmenthttp://en.wikipedia.org/wiki/Sticky_endshttp://en.wikipedia.org/wiki/Ligationhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://c/Documents%20and%20Settings/ANNA.ANNA-257E744327/My%20Documents/Amplified_fragment_length_polymorphism.htmhttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/DNA_fingerprinthttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Polymerase_chain_reaction


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A u&'! o !-' r'!r",!"o*r#+9'*! " !-'* '',!' !o &'#9"/'

T-' #9"/' r#+9'*! #r'"u#"8' o* '*#!ur"*+o4#,r4#9"'+''"!-'r

!-rou+- #u!or#"o+r#-4or


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AFLP%P$R " # -"+-4 '*"!"' 9'!-o or'!',!"*+ o49or-"9"* DNA

T-" !',-*"=u' " ""' "*!o !-r'' !'>

1 D"+'!"o* o !o!# ,'u#r DNA :"!-o*' or 9or' r'!r",!"o* '*849'#*"+#!"o* o r'!r",!"o* -#%"!' ',"/,

##!or !o # r'!r",!"o* r#+9'*!2 S'',!"' #9"/,#!"o* o o9' o !-''r#+9'*! :"!- !:o P$R r"9'r !-#!-#' ,orr'o*"*+ ##!or #*

r'!r",!"o* "!' ',"/, '=u'*,'3 E',!ro-or'!", '#r#!"o* o #9",o*o* # +' 9#!r"?. oo:' &4"u#"#!"o* o !-' * #!!'r*
http://en.wikipedia.org/wiki/Polymorphism_(biology)http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Polymorphism_(biology)


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Step 1: Preparing the AFLPTemplateF"+ur' 1

AFLP #*#4" !#r! :"!- # #9'o +'*o9", DNA "o#!' #*ur"/' ro9 # "*+' "*""u#

A!'r !-' +'*o9", DNA " o&!#"*'."! " "+'!' :"!- # #"r or'!r",!"o* '*849'. o!'* M'I

#* E,oRI (F"+ur' 1) DNA "!4",#4 "*,u!' :"!- !-'''*849' or # 'r"o o*+ '*ou+-!o 'r9"! ,o9'!' "+'!"o* o !-'

DNA "*!o r'!r",!"o* r#+9'*!


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Step 2: Ligation Reaction withRestriction Fragments andAdaptors

I* or'r !o 9#;' u' o !-' r#+9'*!o +'*o9", DNA +'*'r#!' &4 M'I #*E,oRI "+'!"o*. r''#r,-'r *''' #9'!-o !-#! :ou #o: !-'9 !o

+'*'r#!' # u*"=u' !4' o DNA/*+'rr"*! To !-" '*. !-'4 ''o'#* M'I ##!or #* #* E,oRI##!or.


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Step : Selecti!e PCR Amplifcation

A!'r "+#!"*+ !-' +'*o9", DNA

r#+9'*! :"!- !-'"r ,orr'o*"*+##!or. r''#r,-'r #r' '! :"!- #'r"' o DNA r#+9'*! !-#! ,o*"! o!-' ',"/, M'I ##!or%#o,"#!'

'=u'*,'. !-' '!ro4' M'I "!'. #+'*o9", DNA r#+9'*!. !-' '!ro4'E,oRI "!'. #* !-' ',"/, E,oRI##!or%#o,"#!' '=u'*,'


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Step ": #lectrophoreticSeparation o$ Amplifed %&A

Fragments

T-' P$R r'#,!"o* ,#rr"' ou!

ur"*+ !-' !-"r !' o !-' AFLP%P$R ro,' :" "9u!#*'ou4#9"4 # :"' r#*+' o DNA

r#+9'*! "8'. ' o* !-''*+!- o !-' or"+"*# M'I%E,oRI+'*o9", DNA r#+9'*! "+#!' !o!-' M'I #* E,oRI ##!or


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Step ': AnalysisA*#48' !-' r'u!"*+ DNA *"*+

#!!'r* !o '!'r9"*' !-' DNA

/*+'rr"*! #o,"#!' :"!- # +"'*#9' D''*"*+ o* !-',o9'?"!4 o !-' *"*+

#!!'r*. r''#r,-'r ,#* 9#*u#4'!'r9"*' * "8'. or !-'4 ,#*!ur* !o #u!o9#!' #ro#,-'


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T-'r' #r' 9#*4 ##*!#+' !o AFLP :-'*,o9#r' !o o!-'r 9#r;'r !',-*oo+"'"*,u"*+ r#*o94 #9"/' o49or-",DNA (RAPD).

r'!r",!"o* r#+9'*! '*+!- o49or-"9(RFLP). #* 9",ro#!'"!'

AFLP *o! o*4 -# -"+-'r r'rou,"&""!4.r'ou!"o*. #* '*"!""!4 #! !-' :-o'

+'*o9' '' ,o9#r' !o o!-'r!',-*"=u'6@. &u! "! #o -# !-' ,##&""!4!o #9"4 &'!:''* 0 #* 100 r#+9'*!#! o*' !"9'
http://en.wikipedia.org/wiki/RAPDhttp://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphismhttp://en.wikipedia.org/wiki/Microsatellite_(genetics)http://c/Documents%20and%20Settings/ANNA.ANNA-257E744327/My%20Documents/Amplified_fragment_length_polymorphism.htmhttp://c/Documents%20and%20Settings/ANNA.ANNA-257E744327/My%20Documents/Amplified_fragment_length_polymorphism.htmhttp://en.wikipedia.org/wiki/Microsatellite_(genetics)http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphismhttp://en.wikipedia.org/wiki/RAPD


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T-' AFLP !',-*oo+4 -# !-' ,##&""!4 !o'!',! #r"ou o49or-"9 "* "B'r'*!+'*o9", r'+"o* "9u!#*'ou4

AFLP -# &',o9' :"'4 u' or !-'

"'*!"/,#!"o* o +'*'!", #r"#!"o* "* !r#"* or,o'4 r'#!' ',"' o #*!. u*+".#*"9#. #* ,!'r"#

T-' AFLP !',-*oo+4 -# &''* u' "*

,r"9"*# #* #!'r*"!4 !'!. #o !o '!'r9"*'"+-! "B'r'*,' :"!-"* ou#!"o*. #* "*"*;#+' !u"' !o +'*'r#!' 9# or

=u#*!"!#!"' !r#"! o,u(CTL) #*#4"
http://en.wikipedia.org/wiki/Quantitative_trait_locushttp://en.wikipedia.org/wiki/Quantitative_trait_locus


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A NE HIGH%THROUGHPUT AFLP APPROA$HFOR IDENTIFI$ATION OF NE GENETI$

POLYMORPHISM IN GENOME OF THE $LONAL

MI$ROORGANISM MY$OA$TERIUMTUER$ULOSIS


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M4,o,!'r"u9 !u&'r,uo". #,#u' o ''r' "*',!"ou9or&""!4 #* 9or!#"!4 #9o*+

-u9#*. " # ,o*#4 r'rou,"*+"*',!"ou 9",roor+#*"9

T-' 9o! ";'4 '?#*#!"o* or "!

+'*'!", -o9o+'*'"!4 " # -or!'ou!"o*#r4 -"!or4 (K#ur '! #.155@)


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T-" -4o!-'" :# 'r"/' &4 -"+-%!-rou+-u! '=u'*,"*+ o-ou';''"*+ +'*'. :-",-'9o*!r#!' !-#!. '"!' *or9#9u!#!"o* r'=u'*,"' (D#" #*N':9#*.1571).

M !u&'r,uo" o*4 #,,u9u#!'

"9"!' *u9&'r o #'", #r"#*! "*!-'' +'*' (Sr''#!#* '! #. 1557K#ur '! #. 155@ Mu'r '! #.2000)


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Ho:''r. #r"ou #r"#&' ''9'*!or o," :'r' !" "'*!"/#&' "* !-'M !u&'r,uo" +'*o9' T-' /r! "*

!-" 'r"' :# # '! o "*'r!"o*''9'*! "*,u"*+ IS101 ($o"*#* S!'-'*. 1551). IS1@7 (F#*+'! #. 155). #* IS ";' ''9'*!

(M#r"#*" '! #. 1553) #* !-' -"+-4o49or-", IS110 (T-"'rr4 '! #.1550)


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MATERIAL AND METHODS

1.2 Strains and DNA isolationS!r#"* u' or !-' -!AFLP #*#4" -#'

&''* ',r"&' &'or' (Kr'9'r '! #. 1555)

A ur'4 o !-' "o#!' "* !-' ,o',!"o* "+"'* "* T#&' 1 I! " "9or!#*! !o'9-#"' !-#! !:o '#r#!' &u!o'r#"*+ ,o',!"o* o !r#"* :'r' u'T-' /r! ,o',!"o* :# '9o4' or !-'

-!AFLP. :-'r'# !-' ',o* ,o',!"o* :#u' or !-' #"#!"o* o 9#r;'r''o' u"*+ !-' "o! ,o',!"o*


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o!- ,o',!"o* :'r' ro"' "*# &"*' #-"o*. !-' r','""*+"*!"!u!"o* &'"*+ u*#:#r' o !-'

*#!ur' o !-' !r#"* &'or'-!AFLP :# /*"-'

I* !-' #"#!"o* ,o',!"o*. !-r''

!"9' !-r'' ou!&r'#; r'#!'"o#!' :'r' "*,u' !o ,o*/r9"*!'r%!r#"*


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2.2. High-throughput AFLPT-' "*""u# AFLP P$R :'r' 'ror9'

''*!"#4 # '!#"' &'or' "* !-'r''*,' o r#"o#,!"'*u,'o!"' or

!-' "u#"#!"o* o !-' /*+'rr"*! (o'! #. 155) -!AFLP :# 'ror9'u"*+ !-r'' "B'r'*! '*849',o9&"*#!"o* !o "+'! !-' 94,o,!'r"#

DNA> M&oIJT#=I. N#IJT#=I #* M#'IIJN#I(# o'-r"*+'r%M#**-'"9. M#**-'"9.

G'r9#*4)


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E#,- r'!r",!"o* '*849' :# ,o9&"*'

:"!- !-' "+#!"o* o ',"/, "*;'ro"+o*u,'o!"' #"r (M&oI> 5J1> %GTAGA$TG$GTA$$GAT$%3 5J1>GAT$GGTA$G$AGT$TA$%3 N#III>

5J2> %GTAGA$T%G$GTA$A$ATG%35J27> TGTA$G$AGT$T%A$%3 M#'II>51P2> GA$GATGAGT$$TGA$%302K15> $GGT$AGGA$T$AT%3 T#=I>

51P2 #*52H1>AG$$AGT$$TGAGTAG$AG%3)


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For '#,- o !-'' "*;'r ,o9&"*#!"o*.AFLP :# 'ror9' u"*+ '"+-!"B'r'*! "*;'r ',"/, r"9'r,o9&"*#!"o* O*' o !-'' r"9'r:# '?!'*' :"!- # "*+' *u,'o!"'( 1). :-'r'# !-' o!-'r r"9'r :#'=u"' :"!- # 3!'r9"*#

"*u,'o!"' ( 2) T-'' *u,'o!"'ro&' '=u'*,' #r"#!"o* &'4o* !-#!r''*! "* !-' r'!r",!"o* "!' "!'


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T-' '?!'*"o*

:'r' AAJA. AAJG. A$JA. A$JT.AGJA. AGJT. ATJ$ #* ATJGA9"/' 9#!'r"# :# #*#4'

o* 0 20 ,9 o4#,r4#9"'

#&+' #* !-' #9"9'r :'r'"u#"' u"*+ -o-or%"9#+"*+

Po!%AFLP. +' :'r' /?'. r"'#* !or' #! #9&"'*!!'9'r#!ur'


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2.3. Marker selectionse!uencing and geno"icidenti#cation


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F"+ 1 -"+-"+-! !-' '*ro+r#9# ' o* # o !-'' 9#r;'r I! ,#* &'

''* !-#! # o !-' "B'r'*! M !u&'r,uo"+'*o!4' !r#"* (H##r'9. '"j"*+ #* Ar",#)

,u!'r ,o'4 T-' M &o" !r#"* ,u!'r :"!-!-' "*+' M &o" $G #* !-'M4,o,!'r"u9 9",ro!" "o#!'

M4,o,!'r"u9 #r",#*u9 #* M ,#*'!!"" or9=u"!' "!"*,! &r#*,-' "* !-' -4o+'*'!",

!r''. :"!- M ,#*'!!"" o,,u4"*+ !-' 9o!'?,'!"o*# o"!"o* T-' +'*o9' !r#"* :'r'

#*#4' "* u",#!' :"!- !-' +'*o!4"*+r'u! ,u!'r"*+ 9o! ,o'4


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3. $esults

3.1. htAFLP anal%sis and &rag"ent

anal%sis &or M%co'acteriu" strainsT-' /r! 20 M4,o,!'r"u9 !r#"*

("o! ,o',!"o*. '' T#&' 1) :'r'

u&j',!' !o -!AFLP For !-'' !r#"*.!-r'' '*849' ,o9&"*#!"o* :'r',o9&"*' :"!- '"+-! "B'r'*! '',!"'r"9'r #"r !-" +'*'r#!' @0

"B'r'*! /*+'rr"*! !4' or # o !-#!!r#"* T-" r'u!' "* #* o'r#*u9&'r o #ro?"9#!'4 1200#9"9'r


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3.2. DNA se!uencing o& M. tu'erculosisspeci#c

"arkersOu! o !-' o49or-", 9#r;'r

"'*!"/' or M !u&'r,uo". # r#*o94'',!' '! o 20 :# '=u'*,' (T#&' 2).

"*,u"*+ # ',r"!"o* o !-'"r "B'r'*!"#o,,urr'*,' "* !-' AFLP *"*+ #!!'r*

For !-' M !u&'r,uo" 9#r;'r. !-' #!#o&!#"*' :"!- r'',! !o !-' -o9oo+4

'#r,-' or !-' !:o ,urr'*!4 ##"#&'+'*o9' (H37R #* $D 11) #r' =u"!'"9"#r> "* 1 ou! o 20 ,o9#r"o*.-o9oo+ou '=u'*,' :'r' "'*!"/' "*

&o!- +'*o9' '=u'*,'


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3.3. DNA se!uencing o&M%co'acteriu" spp. speci#c

M#r;'r For !-' "*+' !r#"* o

M ,#*'!!"". # 9u!"!u' o9#r;'r :# "'*!"/' &4,#**"*+ /*+'rr"*! T-" " "*u #+r''9'*! :"!- "! ou!4"*+o"!"o* "* !-' o'r# AFLP'*ro+r#9 ('' F"+ 1)


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3.(. Detection o& single nucleotidepol%"orphis" in

"arker &rag"ents &or M. tu'erculosisFr#+9'*! $11. D@ #* H :'r' #9"/'

or # 2 !r#"* "* !-' #"#!"o* ,o',!"o*A9"/,#!"o* :# u,,'u "* # ,#'.

&u! u' o !-' ,orr'o*"*+AFLP r'!r",!"o* '*849' " *o! r'u! "*

"!"*+u"-#&' #!!'r* S'=u'*,"*+ !-''r#+9'*! "* u. -o:''r. r''#' !-'

r''*,' o 12 "*+' *u,'o!"'o49or-"9 (SNP) ('' T#&' @ or !-'r',"' *#!ur' #* o"!"o* o !-'9u!#!"o*)


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Download - Amplified Fragment Length Polymorphism (Aflp)

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