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8/17/2019 Primer Extension, And Nuclease Mapping
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PRIMER EXTENSION AND
NUCLEASE S1 MAPPING.
BIOCHEMISTRY SEMINAR.ALICE MUMBEY-WAFULA
FEB, 20TH 2004.
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PRIMER EXTENSION.
• Th! ! " #$%h&' (h)h $#*+&! %h$ $#$R$/$!$ T"!)*%"!$,(h)h ! "+!& &( "!
RNA-'$*$'$% DNA *&+#$"!$.• R$/$!$ T"!)*%"!$ ("! '!)&/$$' 130 H&("' T$# "' D"/' B"+%#&$
• I% ! &%"$' #"+ 5 %6#&6 )"6!7
/6!$!, +$ A/" M$+&+"!%&!! 86!.• I% )"%"+!$! %h$ !%h$!! &5 )DNA 6!7 " RNA
"! " %$#*+"%$.
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U!$!9
• P#$ $:%$!& ! 6!$' %& #$"!6$ %h$"#&6% &5 " 7/$ RNA "' %& #"* %h$ ;<
$' &5 %h"% RNA.• Th$ )$"%& &5 )DNA +"$! 5
#RNA.
• D$%$)%& &5 %"!)*%! *&'6)$' '67 /%& %"!)*%&.
• M"**7 &5 $*+)"%& %"%& *&%.
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P)*+$ &5 A"+!!
• L$ "++ &%h$ *&+#$"!$!, %h! $#$ $=6$!
&%h " %$#*+"%$ "' " *#$ %& $ $:%$'$'.
• I %h! *&%&)&+, %h$ *#$ $:%$!& ! 6!$' %&#"* %h$ ;
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• Th$ $!6+%"% )DNA 5&#$' ! ""+$' &
" !$=6$)7 7$+.• Th$ +$7%h &5 %h$ $:%$'$' *#$ #"*! %h$
*&!%& &5 %h$ ;< $' &5 %h$ RNA, "' %h$
$+' &5 *#$ $:%$!& *&'6)% $5+$)%!%h$ "6'")$ &5 %h$ RNA.
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M"%$"+!9
A$"+7 #:9
- RNA
- R"'&-+"$+$' &+7&6)+$&%'$! >*#$ (%h
'$5$' !$=6$)$?.
- 0.1 M T!.C+ PH@.
- 0.;M M7.C+2
- RN"!$ 5$ ("%$.
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M"%$"+!9
S%h$!! #:
AM8 $/$!$ %"!)*%"!$
1 M T!.C+ PH @.
0.; M M7.C+2
A)%)4 'NTP #:
RNA!$ 5$$ ("%$
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O%h$ #"%$"+!9
• RNA!$ $")%& #:.
• M !&'6# ")$%"%$
• Ph$&+)h+&&5&#!&"#+ "+)&h&+.
• 100 "' 30 E%h"&+.• L&"'7 '$
• *&+")+"#'$.
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M$%h&'9
1- A$"+7 RNA "' *#$
RNA, '!!&+/$' RNA!$ 5$$ ("%$ !
#:$' (%h "$"+7 655$ )&%"7
&$-$' "'&-+"$+$' *#$ $**$'&5
%6$!. Th$ %6$! "$ )6"%$' 5& 1 #.
" &+7 ("%$ "%h.
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M$%h&'
2- P#$ $:%$!& $")%&
Th$ !%h$!! #: ! %h$ "''$' %& %h$ %6$! "')6"%$' 5& 1 h "% 42&C.
- D$7"'"%& &5 RNA
Th$ RNA!$ $")%& #: ! %h$ "''$' %& $")h *#$$:%$!& %6$, )6"%$' 5& 1; #! "% 3&C. R"!$'$7"'$! %h$ %$#*+"%$ RNA, +$"/7 " )+$"+ +"$+$'!7+$-!%"'$' DNA *&'6)%.
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Th$ $")%&! )"%"+$' $/$!$
%"!)*%"!$.
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Th$ X-"Th$ X-"
S%6)%6$ "'S%6)%6$ "'
R&R&'"7"# &5'"7"# &5
HI8-1 $/$!$HI8-1 $/$!$
T"!)*%"!$.T"!)*%"!$.
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M$%h&', )%'9
• DNA ! %h$ $:%")%$' 5 %h$ *&'6)%
6!7 !&'6# ")$%"%$,
*h$&+,)h+&&5&#!&"#+ "+)&h&+. Th$"=6$&6! +"$ (h)h )&%"! DNA !
%"!5$$' %& " 5$!h %6$.
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M$%h&' >)&%.?
• DNA ! *$)*%"%$' 100 $%h"&+, %h$ *$++$% !
("!h$' 30 $%h"&+ "' " '$' 5& ; %& 10
#. Th$ *$++$% ! $!6!*$'$' +&"'7 7$+,h$"%$' 5& ; # ;&C ("%$ "%h.
• Th$ !"#*+$! "$ +&"'$' *&+")+"#'$3 M
6$" 7$+. Th$ +$7%h &5 %h$ )DNA 7/$! %h$
)*+$%$ #"* &5 %h$ RNA 6* %& ;< $'.
• M"$! &5 &( !$! "' +$7%h "$ +&"'$'
"+&7 !'$ %h$ !"#*+$!.
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Th$ !"#*+$ !
" %&7$%h$ (%h
#"$! &5
&( !$! ,
(h)h h$+*!
%h$ '$%$#"%&
&5 %h$ +$7%h "'
!$.
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P&+")+"#'$ 7$+ $+$)%&*h&$!!, !"#*+$!
1-4 "' #"$! A C
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A'/"%"7$9
N& *&$ ! 6!$', % ! !#*+$ "' !%"7h%
5&("'.
D!"'/"%"7$9
R$/$!$ %"!)*%"!$ !$%#$! $)&6%$!$7&! &5 RNA %h"% )"6!$ % %& *"6!$ &%$#"%$ !%h$!!.
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Th! #&!%+ &))6! G-C )h !%$%)h$!.
I% $!6+%! "'! &5 %$#$'"%$ !$ "'
)&56!$ %h$ %$*$%"%&, "' $'6)$ %h$
$+' &5 56++ $:%$'$' *#$.
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NUCLEASE S1 MAPPING.
B")7&6'9
• I! " !7+$ !%"'- !*$)5) $'&6)+$"!$.
• I% ! !&+"%$' 5 A!*$7++6! &"$"#+"!$.
• I% '7$!%! &%h !7+$-!%"'$' DNA
RNA.• I! " #$%"++&$#$ (%h ) &6' & %&
%! ")%/$ !%$.
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• I! %h$#&!%"+$ $!!%"% %&
'$"%6"%!h7h !"+% )&)$%"%&.
• I% (&! $!% " ")') PH.
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U!$!9
• M"**7 ;< < $'! &5 " %"!)*% 6!7
+"$+$' *&$.
• 6"%%"%7 %h$ +$/$+ &5 " *"%)6+" RNA.
• M"**7 +&)"%&! &5 %&!.
• A"+7 %h$ !%6)%6$ &5 DNA9RNA
h'!.
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U!$!9
• R$#&/7 !7+$-!%"'$' &/$h"7! 5
DNA 5"7#$%! %& *&'6)$ +6% $'!.
• O*$7 %h$ h"* +&&*! 7$$"%$' '67
!%h$!! &5 '!DNA.
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P)*+$ &5 A"+!!9
• H'"%& &5 +"$+$' DNA *&$
5"7#$% %& )$++6+" RNA.
• D7$!%& 6)+$"!$ &5 "++ %h$ !7+$-
!%"'$' $7&!, RNA9DNA h' !
$!!%"% %& %h! )+$"/"7$.
• P&'6)% ! " & " *&+")+"#'$ 7$+.
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• Th$ +"$+$' DNA 5"7#$% %h$ $5+$)%! %h$
"#&6% "' !$ &5 %h$ RNA %h$ h'
%h"% ! h&+&7&6! %& %h$ DNA *&$.
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M"%$"+!9
H'"%& 655$
• M N")+2
• 10 #M EDTA
• @0 #M HEPS PH3.0.
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>)&%.?
H'"%& #:.
• RNA
• L"$+$' &+7&6)+$&%'$ *&$>DNA?
• H'"%& 655$
• T%& X-100• RN"!$ 5$$ ("%$.
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S1 D7$!%& #:.
• 0. M N"C+2• 0 #M N"&A) PH4.
• 2.2#M !&4
• 0.01 T%& X-100
• 20 6%! S1 6)+$"!$
• RN"!$ 5$$ ("%$
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S%&* #:9
• 2.; M N"&A)
• 40 #M EDTA
• 67 %RNA.
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M$%h&'9
• H'"%& &5 RNA 2P +"$+$' DNA *&$.
A++ %6$! #6!% $ RN"!$ 5$$.
• H'"%& #: ! *6% " #)&)$%567$%6$,*+")$' " %h$#"+ ))+$ "' h$"%$' 5&
# "% 4&C >'$"%67?.
• T$#*$"%6$ ! $'6)$' %& ;;&C "' !"#*+$
)6"%$' &/$7h% > "% +$"!% 10 h!.? 5& )*+$%$
h'"%&. T$#*. )" $ "'6!%$' "))&'7 %&
"**+)"%&.
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S1 6)+$"!$ D7$!%&9
S1 6)+$"!$ D7$!%&.
• H'"%& %6$! "$ $#&/$' "' !*"
=6)+, )&%$% %"!5$$' %& 1.; #+$**$'&5 %6$!.
• S1 6)+$"!$ '7$!%& #: ! "''$', #:$'
($++ "' !*".
• T6$! "$ )6"%$' "% 3&C 5& 0 #.
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S%&* $")%&9
• Th$ $")%& ! !%&**$' "''7 !%&* #:.
• S"#*+$! "$ *$)*%"%$' 6!7 100 $%h"&+.
• P$++$% ! ("!h$' 6!7 30 $%h"&+ "' '$' 5&; # " !*$$'/")-$/"*&"%&.
• P$++$% ! $!6!*$'$' TE >T! EDTA? "'
+&"'7 '$>&*h$&+ +6$?.
• T6$! "$ &+$' 5& # "' *+")$' & )$.
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E+$)%&*h&$!!9
• S"#*+$! "$ +&"'$' & " .@ ")+"#'$
7$+ "' !$*""%$' $+$)%&*h&$!!.
• L"$+$' #"$! "$ +&"'$' "+&7 (%h %h$!"#*+$!.
• Th$ 7$+ ! '$' "' $:*&!$' %& X-" 5+#.
• R$!6+%! "$ $"' '$)%+ 5 %h$
"6%&"'&7"#.
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A $:"#*+$A $:"#*+$
O5 " "6%&"-O5 " "6%&"-
'&7"*h,'&7"*h,Sh&(7 "'!Sh&(7 "'!
(%h(%h 22PP..
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D!"'/"%"7$ &5 ""+!!.
• D$*6"%& #" &))6 '6$ %& +&( PH "%
(h)h 6)+$"!$ (&!. Th! +#%! %!"**+)"%&!.
• I5 h7h )&)$%"%& ! 6!$', " $" )" $
)$"%$' %h$ DNA '6*+$:.• U!$ &5 +"$+$' *&$ #"$! % "
)*+)"%$' *&)$'6$.
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R$5$$)$!9
• 8&$% D 8&$% J. B&)h$#!%
• A. K&$7 "' B"$. DNA $*+)"%&.
• C6$% *&%&)&+! #&+$)6+" &+&7.W+$ "' !&!.
• h%%*9(((.#(2.%$!)$)$.(+$.))*
&+$%!$/$.• h%%*9(((.5h)).&7+"!h"h#$%h&'!#&
+-&-#$%h.