More on Nested PCR

PCR: Yield of specific and nonspecific products! ! !"#$%&'$(&)#*"&#+,-#.'*%/'01#2$#.'*%/'01#/1#34&#%/'$5#(&"*6/7#89:#/"3&('$3&;#/"3*#34&#4*13#(&"*6&<#/1#

as a function of template levels! =*0";#/"#&%&'>#?@)@@@#A5**;#7&551#='*6#$"#/"=&73&;#.&'1*")#34*0(4#34/1#7$"#%$'>#BC#*';&'1#*=#
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! +06$"#(&"*6&#F#I)H@@)@@@)@@@#A.
! +,-#89:#/1#GEH#J?@KLIEH#J?@G#M#?@H#F#?#.$'3#/"#IEN#A/55/*"
! :#?O#"3#.'/6&'#P*05;#A&#3**#14*'3)#A03#/=#$"#+,-#.'/6&'#/1#C@QCO#"3#/"#5&"(34#/3#P/55#.'*A$A5>#A&#
0"/R0&#$";#"*#&J$73#6$374#P*05;#A&#=*0";#/"#34&#406$"#(&"*6&)#$1106/"(#34$3#34&#406$"#
(&"*6&#1&R0&"7&#P$1#&11&"D$55>#'$";*6S#2H<?O#F#?J?@G)#2H<C@#F#?J?@?C)#2H<CO#F#?J?@?O
! +*P&%&')#34&'&#$'&#&";*(&"*01#406$"#'&3'*%/'01Q5/T&#1&R0&"7&1#34$3#6/(43#4$%&#.$'D$5#
4*6*5*(>#3*#+,-#2$";#P*05;#.'*;07&#34&#1$6&#1/U&;#A$";1<)#:98#34&#KV#&";#A$1&1#*=#34&#
.'/6&'#$'&#=$'#6*'&#/6.*'3$"3#3*#34&#1.&7/W7/3>#*=#34&#.'/6&'#34$3#34&#'&6$/"/"(#A$1&1
! ,"#1066$'>)#/3#/1#;/X7053#3*#YZ[#$6.5/=>#+,-S
! \4&'&#/1#$#3'&6&";*01#&J7&11#*=#7*6.&D"(#1&R0&"7&1
! \4&'&#$'&#.$'D$55>#4*6*5*(*01#1&R0&"7&1#/"#34&#406$"#(&"*6&#Q#;0&#3*#&";*(&"*01#%/'01Q
5/T&#1&R0&"7&1#$";#KV#&";#4*6*5*(>#&J.&73&;#*"#$#'$";*6#A$1/1

15,! 150! 1.5! 15,! 150! 1.5! 15,! 150! 1.5! ! \4&#'&1053#/1#34$3#P4/5&#+,-#1&R0&"7&1#;*#A&7*6&#&"'/74&;#$1#$#'&1053#*=#34&#W'13#'*0";#*=#YZ[)#6074#
*=#34&#$6.5/W&;#6$3&'/$5#=*55*P/"(#$#3>./7$5#KO#7>75&1#*=#YZ[#/1#"*3#+,-
?! 000! 1,! 15! 150,! 000! 1,! 15! 150,! 000! 1,! 15!
500! 000! 500! 000! 500! ! 9&13&;#YZ[#*%&'7*6&1#6$">#*=#34&1&#.'*A5&61#A>#.'*%/;/"(#$#"&P#1&3#*=#.'/6&'1#34$3#A/";#3*#+,-#$3#
;/]&'&"3)#/"3&'"$5#1/3&1#P/34/"#34&#;&1/'&;#$6.5/7*"
Number of template molecules! ! \4&#5/T&5/4**;#34$3#$#7&5505$'#1&R0&"7&#P*05;#.*11&11#10X7/&"3#4*6*5*(>#3*#$55#H#.'/6&'#A/";/"(#
'&(/*"1#/1#6074#5*P&'#34$"#=*'#C#.'/6&'#1&R0&"7&1
present within 150,000 cells worth of DNA ! \4&#"&13&;#'*0";#34&")#&11&"D$55>#'&13*'&1#34&#1.&7/W7/3>#*=#$6.5/W7$D*"#

Nested PCR greatly improves yield and specificity!

Primer Promiscuity!

Relative efficiency w/ 3' terminal mismatches:!

!
! ! !Primer 3' base!
! ! !T !C !G !A!
Template !T !1 !1 !1 !1!
3' base ! !C !1 !<0.01 !1 !1!
! !G !1 !1 !1 !<0.01!
! !A !1 !1 !<0.01 !0.05!
!
 Hemi-Nested PCR
(One new primer in PCR
second round of PCR)
Hemi-nested sensitivity.
Nested PCR

Maeda H et al. Chest 2000;118:923-927

2000 by American College of Chest Physicians