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1993 3: S30-S37Genome Res.C W Dieffenbach, T M Lowe and G S Dveksler
General concepts for PCR primer design.
Referenceshttp://genome.cshlp.org/content/3/3/S30.refs.html
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General
oncepts for
P R Prim er
Design
C.W. Dief fenbach 1
T.M.J. Lowe 2 and
G.S. Dve ksler ~
1Division of AIDS, Nat iona l
Institute of Allergy and Infectious
Diseases, National Institutes of
Health, Bethesda, Maryland 20892;
ZDepartment of Mo lecular Biology,
Washington University, St. Louis,
Missouri 63110; 3Department of
Pathology, Uniformed Services
University of the Health Sciences,
Bethesda, Maryland 20814
P C R i s a te c h n o l o g y b o r n o f t h e m o d e r n m o l e c u l a r b i o l o g y e r a. T h e e n z y m e
used for PCR,
Taq D N A p o l y me r a s e , s u p p l i e d w i t h t h e 1 0 x b u f f e r , is p u r -
c h a s e d a s a c l o n e d p r o d u c t , a n d t h e n u c l e o s i d e t r i p h o s p h a t e s a r e u l t r a p u r e ,
b u f f e r e d , a n d a v a i l a bl e a t a c o n v e n i e n t c o n c e n t r a t i o n . Y et , w i t h a l l o f t h e s e
commerc ia l ly ava i l ab le s t a r t ing mater i a l s , PCR s t i l l fa i l s , par t i cu lar ly fo r the
n o v i c e . A s s u mi n g t h a t a l l o f t h e r e a g e n t s h a v e b e e n a d d e d i n t h e p r o p e r
c o n c e n t r a t i o n s , t w o c r i ti c a l PCR c o m p o n e n t s a r e l e ft t o t h e r e s e a r c h e r . T h e
f i r s t i s t h e n u c l e i c a c i d t e mp l a t e , w h i c h s h o u l d b e o f s u f f i c i e n t q u a l i t y a n d
c o n t a i n n o i n h i b i t o r s o f
Taq
D N A p o l y m e r a s e ( a l t h o u g h w h e n i t c o m e s t o
t e m p l a t e p u r i t y, P C R is m o r e p e r m i s s iv e t h a n m a n y o t h e r m o l e c u l a r b i o l o g y
t e c h n i q u e s ) . T h e s e c o n d i s t h e s e l e c t i o n o f t h e o l i g o n u c l e o t i d e p r i me r s . T h i s
p rocess i s o f t en cr i t i ca l fo r the overa l l success o f a PCR exper iment , fo r wi th -
o u t a f u n c t i o n a l p r i me r s e t, t h e r e w i l l b e n o PCR p r o d u c t . A l t h o u g h t h e
s e l e c t i o n o f a s in g l e p r i me r s e t ma y b e t r iv i a l, t h e c o n s t r u c t i o n o f p r i me r s e ts
f o r a p p l i c a t i o n s s u c h a s mu l t i p l e x o r n e s t e d PCR b e c o me s mo r e c h a l l e n g i n g .
T h e ma n u a l s e l e c t i o n o f o p t i m a l PCR o l i g o n u c l e o t i d e p r i me r s e ts c a n b e
q u i t e t e d i o u s a n d t h u s l e n d s i t se l f v e r y n a t u r a l l y t o c o mp u t e r a n a l y s is . T h e
p r i m a r y f a c to r s t h a t a ff e c t t h e f u n c t i o n o f t h e o l i g o n u c l e o t i d e s - - t h e i r me l t -
i n g t e m p e r a t u r e s a s w e l l a s p o s s i b l e h o m o l o g y a m o n g p r i m e r s - - a r e w e l l -
d e f i n e d a n d s t r a i g h t f o r w a r d t a s k s t h a t a r e e a s i ly e n c o d e d i n c o m p u t e r s o f t -
w a r e. O n c e t h e c o m p u t e r h a s p r o v i d e d a sm a l l n u m b e r o f c a n d i d a t e p r i m e r
se t s , t he t ask o f se lec t ion can be (and s t i l l i s ) per fo rmed manual ly . In th i s
a p p r o a c h , t h e r e s e a r c h e r i s t a k i n g a d v a n t a g e o f t h e r a w s p e e d o f c o m p u t e r
c a l c u l a t io n s , t r y i n g a ll p o s s i b l e p e r m u t a t i o n s o f a p r i me r ' s p l a c e me n t , l e n g t h ,
a n d r e l a t i o n t o t h e o t h e r p r i me r s t h a t me e t c o n d i t i o n s s p e c i f i e d b y t h e u s e r .
F r o m t h e t h o u s a n d s o f c o m b i n a t i o n s t e st e d b y t h e c o m p u t e r , a so f tw a r e
p r o g r a m c a n p r e s e n t j u st t h o s e t h a t a r e s u i ta b l e f o r t h e n e e d s o f t h e e x p e r i -
me n t . T h u s , t h e o v e r a l l q u a l i t y ( as d e f i n e d b y t h e u s e r i n p r o g r a m p a r a m-
e t e r s ) o f t h e p r i me r s s e l e c t e d i s a l mo s t g u a r a n t e e d t o b e b e t t e r t h a n t h e
h a n d f u l c h o s e n a n d h a n d - t e s t e d b y th e r e s e a rc h w i t h o u t c o m p u t e r a ss is -
t ance .
A s w i t h a n y t o o l , u n d e r s t a n d i n g i ts f u n c t i o n w i l l m a k e t h e e n d p r o d u c t
m o r e u s ef u l. A w i d e r a n g e o f p r o g r a m s h a v e b e e n w r i t t e n t o p e r f o r m p r i m e r
s e l e c t i o n , v a r y i n g s i g n i f i c a n t l y i n s e l e c t i o n c r i t e r i a , c o mp r e h e n s i v e n e s s , i n -
t e rac t ive des ign , and user - f r i end l ine ss . (1-1~ There a re a l so co m me rc i a l ly
a v a i l a b l e s p e c i a l t y p r i me r d e s i g n s o f t w a r e p r o g r a ms t h a t o f f e r e n h a n c e d u s e r
in ter faces , add i t iona l fea tu res , a nd up dat ed se lec t ion cr i t e r ia , (1'2 ) as wel l as
p r i me r d e s i g n o p t i o n s t h a t h a v e b e e n a d d e d t o l a r g e r , mo r e g e n e r a l s o f t w a r e
packages .
A l t h o u g h m o s t p e o p l e w o u l d a g r e e t h a t a p p l i c a t i o n o f a n a l y t i c c o m p u t e r
s o f t w a r e t o a w e l l - d e f i n e d p r o b l e m i s a s ma r t t h i n g t o d o , n o t a l l r e s e a r c h e r s
a r e c o n v i n c e d t h a t PCR p r i me r s e l e c t i o n i s a n o n t r i v i a l t a s k, o r t h a t t h e
s e l e c t i o n r u l e s t h a t ma k e a p r i me r a mp l i f y e f f i c i e n t l y a r e e v e n w e l l d e f i n e d .
E v e n t h o u g h ma n y o f t h e r u l e s d i s c u s s e d h a v e b e e n f i n e - t u n e d b y c o l l e c t i v e
e m p i r i c a l w is d o m , m o s t a re b a se d o n f i rm t h e o r e ti c a l g r o u n d , i f n o t c o m m o n
s e n se . T h e p u r p o s e o f t h i s c h a p t e r i s t o e x p l a i n b a s i c r u l e s o f o l i g o n u c l e o t i d e
p r i m e r d e s i g n . W i t h t h i s u n d e r s t a n d i n g o f p r i me r s e l e c t i o n c r it e r ia , t h e i n -
f o r m a t i o n d e d u c e d b y p r i m e r d e s i g n s o f t w a r e c a n b e r a t i o n a l l y i n t e r p r e t e d
a n d m a n i p u l a t e d t o fi t y o u r e x p e r i m e n t a l n e e d s .
P R METERS USED IN B SIC PCR PRIME R DESIGN
Pr i me r d e s i g n i s a i me d a t o b t a i n i n g a b a l a n c e b e t w e e n t w o g o a l s : s p e c i f i c i t y
a n d e f f i c i e n c y o f a mp l i f i c a t i o n .
pecificity
s d e f i n e d a s t h e f r e q u e n c y w i t h
w h i c h a mi s p r i mi n g e v e n t o c c u r s . P r i me r s w i t h me d i o c r e t o p o o r s p e c i f i c i t y
t e n d t o p r o d u c e P C R p r o d u c t s w i t h e x t ra u n r e l a t e d a n d u n d e s i r a b l e a m p l i -
3 0
P R
Me t h o d s a n d A p p l i c a t i o n s
3:$30-S379 by Cold Spring Harbor Laboratory ISSN 1054-9805/93 $1.0
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cons as visualized on an ethidium bromide-stained agarose gel. fficiency is
defined as how close a primer pair is able to amplify a product to the theo-
retical optimum of a twofold increase of product for each PCR cycle.
Given a target DNA sequence, analysis software attempts to strike a balance
between these two goals by using preselected default values for each of the
primer design variables. These variables, listed below, have predictable effects
on the specificity and efficiency of amplification. Depending on the experi-
menta l requirements, these prime r search paramete rs can be adjusted to
override the default values that are mean t to be effective for only general PCR
applications. For example, in medical diagnostic PCR applications, search
parameters and reaction conditions would be adjusted to increase specificity
at the cost of some efficiency, because avoiding false-positive results is a
higher priority in this case than producing large quantities of amplified prod-
uct. By carefully considering the following parameters when using primer
design software, more effective selection of primers will be achieved.
Primer ength
The specificity is generally controlled by the length of the primer and the
annealing temperature of the PCR reaction. Oligonucleotides between 18 and
24 bases tend to be very sequence specific if the a nnea ling te mperat ure of the
PCR reaction is set within a few degrees of the primer Trn (defined as the
dissociation temperature of the primer/template duplex). These types of oli-
gonucleot ides work very well for standard PCR of defined targets that do n ot
have any sequence variation. The longer the primer, the smaller the fraction
of primed templates there will be in the annealing step of the amplification.
In exponential amplification, even a small inefficiency at each annealing step
will propagate to produce a significant decrease in amplified product. In
summary , to optimiz e PCR, the utilizatio n of primers of a min ima l l engt h
that ensures melting temperatures of 54~ or higher will provide the best
chance for maintenance of specificity and efficiency.
Short oligonucleotides of 15 bases or less are useful only for a limited
am ou nt of PCR protocols such as the use of arbitrary or ran dom short p rimers
in mappi ng simple genomes and in the subtraction library protocol described
by Willi ams and Liang and Pardee. (11'12) Dep endin g on t he geno me size of
the organism, there is a mi ni mu m pr imer length . In general, it is best to build
in a marg in of specificity for safety. For each a dditiona l nucleoti de, a primer
becomes four times more specific; thus, the minimum primer length used in
most applications is 18 nucleotides. Clearly, if purified cDNA is being used, or
genomic DNA is not present, the length could be reduced because the risk of
nonspecific primer/template interactions will be greatly reduced. Yet, it is
generally a good idea to design primers such that the synthesized oligonu-
cleotides can be used in a variety of experiment al c onditi ons (18- to 24-mers),
and the small marginal cost of oligonucleotides with four to five additional
bases makes it worth the expense.
The upper limit on primer length is somewhat less critical and has more to
do with reaction efficiency. For entropic reasons, the shorter the primer, the
more quickly it will anneal to target DNA and form a stable double-stranded
template to which DNA polymerase can bind. In general, oligonucleotide
primers 28--35 bases long are necessary when amplifying sequences where a
degree of heterogeneit y is expected. This has proved to be generally useful i n
two types of applications: (1) In amplifying sequences encoding closely re-
lated molecules, such as isoforms of a protein or family of proteins within a
species, as well as in the cloning of the homologous gene from a different
species, O3) and (2) in ampl ifying the sequences of viruses such as HIV-1,
where the possibility of having a set of primers with perfect complementary
PCR M e t h o d s a n d A p p l i c a t io n s 3 1
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to a l l the t em pla tes ( in th is exa mp le , a l l HIV-1 i so la te s) i s not exp ec ted . (14,1s)
I n bo th c a se s , one f i rs t u se s p r im e r de s ign so f tw a r e t o c om p a r e a l l a va i l a b l e
r e l a t ed s e q u e n c e s a n d , i n s u c h a m a n n e r , d e t e r m i n e s t h e D N A r e g i o n w i t h t h e
l e a s t a m oun t o f s e que nc e va r i a b i l i t y . T he se r e g ions s e r ve a s s t a r t i ng p l a c e s f o r
s e l e c t in g t h e p r i m e rs . I n s o m e i n s t a n c e s , t h e r e s e a r c h e r a lr e a d y k n o w s t h e
f u n c t i o n o f t h e e n c o d e d p r o t e i n a n d t h e d o m a i n s e s s en t i al t o p e r f o r m i n g t h a t
f u n c t i o n . I n t h e s e ca s es , c o m p a r i n g a v a i la b l e s e q u e n c e s i n t h e r e g i o n s c r i ti c a l
f o r t h e f u n c t i o n a l a c t i vi t y of t h e r e l a t e d p r o t e i n s w i t h i n t h e f a m i l y w i ll a i d i n
d e f i n i n g t h e s e q u e n c e s f o r d e s i g n i n g n e w p r i m e r s . E x a m p l e s i n c l u d e t h e P C R
c l o n i n g o f a n e n z y m e o r r e c e p t o r w i t h a s i m i l a r s t r u c t u r e a n d f u n c t i o n f r o m
a r e l a t e d s p e c i e s u s i n g t h e a v a i l a b l e s t r u c t u r a l d a t a . W i t h t h e a m i n o a c i d
s e q u e n c e i n f o r m a t i o n a n d t h e h e l p o f c o d o n u s a g e t a b l e s f or d i f fe r e n t s p e -
c ie s, b o t h p r i m e r s , o r a t l e as t o n e o f t h e m , c o u l d b e d e s i g n e d a r o u n d t h e
c o n s e r v e d s e q u e n c e . W h e n s e l e c t i n g p r i m e r s t o a m p l i f y D N A f r o m a d if -
f e r e n t s p e c i e s , s e q u e n c e s a t t h e 5 ' - o r 3 ' - u n t r a n s l a t e d r e g i o n s o f t h e m R N A
s h o u l d b e a v o i d e d b e c a u s e t h e y m a y n o t n e c e s s a r i ly h a v e h i g h d e g r e e o f
h o m o l o g y .
T he p l a c e m e n t o f t he 3 ' e nd o f t he p r im e r i s c r i t i c a l f o r a suc c e s s f u l P C R
r e a c t ion . I f a c onse r ve d a m i no a c id c a n be de f ine d , t h e f i r s t 2 ba se s o f t he
c o d o n , o r 3 b a s es i n t h e c a s e o f a n a m i n o a c i d e n c o d e d b y a s in g l e c o d o n
( m e t h i o n i n e a n d t r y p t o p h a n ) , c a n s e rv e a s t h e 3 ' e n d . P e r f ec t b a s e - p a i r i n g
b e t w e e n t h e 3 ' e n d o f t h e p r i m e r a n d t h e t e m p l a t e i s o p t i m a l f o r o b t a i n i n g
g o o d r e su l ts ; m i n i m a l m i s m a t c h s h o u l d e x i st w i t h i n t h e l a st 5 t o 6 n u c l e -
o t i d e s at t h e 3 ' e n d o f t h e p r i m e r. A t t e m p t s t o c o m p e n s a t e f o r t h e m i s m a t c h e s
b e t w e e n t h e 3 ' e n d o f t h e p r i m e r a n d t h e t e m p l a t e b y l o w e ri n g t h e a n n e a l i n g
t e m p e r a t u r e o f t h e r e a c t i o n s d o n o t i m p r o v e t h e r e s u lt s , a n d f a i l u re o f t h e
r e a c t i o n is a l m o s t g u a r a n t e e d . W i t h t h i s c o n c e p t i n m i n d , o n e s h o u l d e v a l -
u a t e a l l p o s s i b l e s t r a t e g i e s i n t h e d e s i g n o f p r i m e r s w h e n t h e n u c l e o t i d e
s e q u e n c e o f t h e t e m p l a t e t o b e a m p l i f i e d i s n o t k n o w n w i t h c e r t a i n ty . C a s e s
l i k e t h e o n e d e s c r i b e d a b o v e a r e r o u t i n e l y e n c o u n t e r e d w h e n t h e r e s e a r c h e r
w i s h e s t o a m p l i f y a cD N A u s i n g i n f o r m a t i o n f r o m a p ar t i al p r o t e i n s e-
q u e n c e . (13) S e v er a l a p p r o a c h e s t h a t i n c l u d e t h e u s e o f d e g e n e r a t e o l i g o n u c l e -
o t i d e p r i m e r s c o v e r i n g a ll po s s i b le c o m b i n a t i o n s f o r t h e b a s e s a t t h e 3 ' e n d o f
the p r im e r i n t he poo l , a s w e l l a s t he u se o f i nos ine t o r e p l a c e t he ba se
c o r r e s p o n d i n g t o t h e t h i r d o r v a r i a b l e p o s i t i o n o f c e r t a i n a m i n o a c i d c o n -
d o n s , h a v e b e e n s u c c es s f u l f o r c D N A c l o n i n g a n d f o r d e t e c t i o n o f s e q u e n c e s
w i th pos s ib l e va r i a t i ons . (16) M u c h o f t h i s t ype o f P C R s tudy i s e m p i r i c a l , a nd
d i f f e r e n t p r i m e r s m a y h a v e t o b e s y n t h e s i z e d t o o b t a i n t h e d e s i r e d m a t c h .
L o n g e r p r im e r s c o u l d a l s o ar i se w h e n e x t ra s e q u e n c e i n f o r m a t i o n , s u c h a s
T 7 R NA po ly m e r a se - b ind i ng s i te , r e s t r i c t i on s i t e s, o r G C c l a m p , i s a dd e d to
p r im e r s . (17-19) I n ge ne r a l , t he a dd i t i on o f un r e l a t e d s e qu e nc e s a t t he 5 ' e n d o f
t h e p r i m e r d o e s n o t a l te r t h e a n n e a l i n g o f t h e s e q u e n c e - s p e c i f i c p o r t i o n o f
t h e p r i m e r . I n s o m e c a s es , w h e n a s i g n i f i c a n t n u m b e r o f b a s e s t h a t d o n o t
m a t c h t h e t e m p l a t e s e q u e n c e a r e a d d e d t o t h e p r i m e r , f o u r t o f i v e c y c l e s o f
a m p l i f i c a t i o n c a n b e p e r f o r m e d a t a l o w e r a n n e a l i n g t e m p e r a t u r e f o l l o w e d b y
t h e r e s t o f t h e c y c l e s a t t h e a n n e a l i n g t e m p e r a t u r e , c a l c u l a t e d w i t h t h e a s -
s u m p t i o n t h a t t h e s e q u e n c e a t t h e 5 ' e n d o f t h e p r i m e r i s a l r e a d y i n c o r p o -
r a t e d i n t o t h e t e m p l a t e . A d d i t i o n a l b a s e s a t t h e 5 ' e n d o f t h e p r i m e r s a r e
f r e q u e n t l y a d d e d w h e n t h e r e s e a r c h e r n e e d s t o c l o n e t h e P C R p r o d u c t . I n
t h e s e c a se s, t h e r e s t r i c t io n e n z y m e s i te s of c h o i c e w i ll b e t h e o n e s t h a t d o n o t
c u t w i t h i n t h e D N A a t s i t e s o t h e r t h a n t h e p r i m e r . T o e n s u r e s u b c l o n i n g o f
t h e w h o l e a m p l i f i e d f r a g m e n t o f u n k n o w n s e q u e n c e a s a s i n g l e p i e c e, a d d i -
t i o n o f s i t e s f o r e n z y m e s t h a t r e c o g n i z e 6 b a s e s o r t h e a d d i t i o n o f p a r t i a l l y
o v e r l a p p i n g r e c o g n i t i o n s it e s f o r d i f f e re n t e n z y m e s i s r e c o m m e n d e d . A n i m -
p o r t a n t c o n s i d e r a t i o n w h e n a d d i n g r e s t r ic t i o n s i te s t o a p r i m e r i s t h e f a ct t h a t
m o s t e n z y m e s r e q u i r e t w o o r t h r e e n o n s p e c i f i c e x t r a b a s e s 5 ' t o t h e i r r e c o g -
32
P R M etho ds
and pp l i c a t i o n s
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nition sequence to cut efficiently, adding to the length of the nontemplate-
specific porti on of the primer. (2~ Anothe r drawbac k of long pr imer sequences
is in the calculation of an accurate melting temp eratur e necessary to establish
the annealing temperature at which the PCR reaction is to be performed. For
primers shorter than 20 bases, an estimate of Tm can be calculated as Tm = 4
(G + C) + 2 (A + T), (21) whereas for lon ger pr imers the Tm requ ires the nearest-
neighbor calculation, which takes into account thermodynamic parameters
and is employed by most of the available computer programs for the design
of PCR primers. (22'23)
The Termi nal Nucleot ide in the PCR Prim er
Kwok and colleagues have shown that the 3'-terminal position in the primer
is essential for cont rol ling misp riming. (24) For certain appl icati ons as de-
scribed above, this chance of mispriming is useful. The other issue concern-
ing the 3' ends of the PCR primers is the prevention of homologies within a
primer pair. Care has to be taken that the primers are not complementary to
each other, particularly at their 3' ends. Complementarity between primers
leads to the undesirable primer- dimer p hen ome na in which the PCR product
obtaine d is the result of the a mplification of the primers themselves. This sets
up a competitive PCR situation between the primer-dimer product and the
native template and is detrimental to the success of the amplification. In cases
when multiple primer pairs are added in the same reaction (multiplex PCR),
it is very important to check for possible complementarity of all the primers
added in the reaction. Generally, the computer programs do not allow primer
pairs with 3'-end homologies and, in conjunction with the hot start tech-
nique, the chances of formation of primer-dimer products are greatly re-
duced. (25)
Reasonable GC Content and T.
PCR primers should maintain a reasonable GC content. Oligonucleotides 20
bases long with a 50 G + C conte nt gen erally have Tm values in the range of
56-62~ This provides a sufficient therma l window for efficient annea ling.
Within a primer pair, the GC content and Tm should be well matched. Poorly
matched primer pairs can be less efficient and specific because loss of spec-
ificity arises with a lower Tm and the primer with th e hi gher Tm has a greater
chance of mispriming under these conditions. If too high a temperature is
used, the primer of the pair with the lower Tm may not function at all. This
matching of GC content and Tm is critical when selecting a new pair of
primers from a list of already synthesized oligonucleo tides with in a sequence
of interest for a new application. For this reason we advocate the adop tion of
a standardized criteria for primer selection within a laboratory. By planning
ahead, it is easier to mix and match selected primers, as they will all have
similar physical characteristics.
PCR Product Length and Placement within the Target Sequence
All of the computer programs provide a place for selecting a range for the
length of the PCR product. In general, the length of the PCR product has an
impa ct o n t he efficiency of amplification . (26) The l eng th of a PCR pro duc t for
a specific application is dependent in part on the template material. Clinical
specimens pre pared from fixed tissue samples tend to yield DNA that does n ot
support the amplificatio n of large products. (27) From pure plasmid or h igh-
molecula r-weight DNA, it is relatively straightforward to obt ain products >3
kb. For the purpose of detecting a DNA sequence, generally PCR products of
150-1000 bp are produced. The specifics of the size of the desired products
PCR Me t h o d s a n d p p l i c a ti o n s 33
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often depend on the application. If the purpose is to develop a clinical assay
to detect a specific DNA fragment, a small DNA amplification product of
120-300 bp may be optimal. The product should be specific and efficient to
produce while containing enough information for use in a capture probe
hyb ridi zat ion assay. ~28) Products in this size range can be produc ed usi ng t he
two-step amplification cycling method, thereby shorte ning the length of the
amplification procedure. Other PCR approaches have different optimal prod-
uct lengths. For example, for the purpose of monitoring gene expression by
quantita tive RNA PCR, the pr oduct must be large enou gh that a competitive
template can be constructed and both can be easily resolved on a gel. These
products generally are in the 250- to 750-bp range. Here, the issue is maxi-
mizing the efficiency of both the reverse transcriptase step and the PCR. In
terms of placemen t of the PCR primers wi thin a cDNA sequence, two specific
points should be kept in mind. The first is to try to keep the primers and
product within the coding region of the mRNA. This is the unique sequence
that is responsible for the production of the protein, unlike the 3 - nonc odin g
region that will share homologies with many different mRNAs. The second
point is to try to place the primers on different exons. In so doing, the
RNA-specific PCR product will be different in size from one arising from
con tami nati ng DNA. If the purpose of the PCR is to clone a specific region of
a gene or cDNA, then the size of the PCR product is preselected by the ap-
plication. Here, the computer program can provide information about se-
lected primer sets that flank the desired area. In some instances, when the
complete sequence is required for further experiments and the PCR product
to be obtained is above the ideal length, or the template is not of the best
quality, overlapping PCR fragments can be amplified by designing primers
flanking unique restriction sites in the template sequence. The production of
a fragment containing the entire sequence will then be obtained by cutting
and pasting the amplified pieces. When approaching this kind of application
it is important beforehand to think about the ideal method for cloning the
PCR products and how the clone will be used in the future. For example, if
one wishes to utilize restriction endonuclease sites at the end of primers as
described above, it is important to be sure that these enzymes do not cut
within the amplified region. Software programs can provide this informa-
tion. (2)
A Simple Rule for Noncomputer based Selection
Occasionally, the PCR primers must be selected from very defined regions at
the 3 and 5 ends of a specific sequence. A simple met hod of prim er design
is to choose regions that are deficient in a single nucleotide. By selecting
primers in this way, the chance of extensive primer-primer homology is re-
duced. Here, again, care must be taken to have a balanced primer pair in
terms of length and base composition so that the Tm of the p rimers are well
matched.
Nested PCR
In certain situations, there are unresolvable problems with the quantity and
quality of the template to be amplified. Perhaps the actual quantity of target
nucleic acid is very dilute relative the rest of the material present, or there is
a limit on the purity of the starting material. In certain clinical applications,
both of these prob lems occur simultan eously. (29) In these circumstances, one
approa ch to synthe sizing a product reliably is to develop a nested PCR assay.
Generally, the sample is first amplified for 20-30 cycles using the outer
primer set; a very small aliquot of this reaction is then amplified a second
time for 15-25 cycles using the inner primer set. The in ner set of PCR primers
3 4 PCR M e t h o d s a n d A p p l i c a t i o n s
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is positioned within the DNA so that the complementary sequence for the
inner primer pair is present in the PCR product obtained in the first ampli-
fication reaction and available to form a template/prime r complex. This has
been shown to be more successful than diluting and reamplifying with the
same primers. (29) The positi on of the inn er pr imer set is often t he det ermi n-
ing factor in the overall structure of the nested approach and will be a factor
in determining the final product size. For example, in the nested PCR detec-
tion system, adapted from t he or iginal assay of Larder et al. (3~ for the ami no
acid 215 mu tat ion in the H IV-1 reverse transcrip tase involved i n azi dothy mi-
dine resistance, the 3 end of one of the inner primers must matc h the mu-
tation. (3~ A contro l inn er p rimer set, ru n in parallel, detects t he wi ld-type
sequence. In general, the product of the inner set is small, 120-270 bp. When
selecting nested primer sets, special care must be given to eliminate potential
primer dimers and matches between members of the inner and outer primer
sets. Some of the software programs for primer design have the selection of
nested primers as an option.
U TIL IZ IN G P R IME R D E SIGN S OFTW R E
It is important to stress that the primer selection parameters described here
are general and are not necessarily implemented in the same manner among
the different primer selection software. Thus, two programs using slightly
different selection algorithms will rarely, if ever, select the exact same
primers, ev en if the basic parameters are equi valent ly set. These discrepancies
are attributable to differences in the calculation methods and the order in
which the selection criteria are applied. For example, calculating the temper-
ature of primer/template annealing can be performed in one of several
ways. The origi nal formula of Suggs and co-workers, ~21~ Tm =2 ~ +
4~215 G + C), is popular for its simplicity and rough ly accurate p redi ctio n of
oligonucleotide Tm. More recentl y, Rhylick et al. ~26~ im pl em en te d Tm predic-
tion based on neares t-ne ighbo r t her mod yna mi c parameter s, ~22 23~ whi ch ap-
pear to be slightly more accurate. Other programs base primer annealing
temperatures on formulas originally developed for DNA fragments ~ 100 nu-
cleotides long. ~31~ Thus, specifying a desired p rime r ann eal ing tem per atu re to
be 60~ will produce different primers from th e exact same target sequence.
Further work by Rychlik and co-workers ~26~ prod uced an empiri cally derived
equation for the optimal an nealing tempera ture of a primer pair that depends
on near est n eighb or calcul ation s. Wu et al. ~32~ have also empi ric ally derived
an equation, based on primer length and GC content, to determine optimal
oligonucleotide annea ling temperature. These examples illustrate how some-
thing as basic as primer Tm calculation can vary among the programs.
Second, different programs attack the task of primer selection very differ-
ently, applying selection criteria to reduce the number of possible primers
that the program must consider while not eliminating potentially good can-
didates. For example, t he progr am by Lowe and colleagues (2) only considers
primers tha t have a 3 -end CC, GG, CG, or GC dinucleotide, whi ch may
increase priming efficiency while allowing the user to specify a range of
prim er lengths. In contrast, the progr am by Rychlik and Rhoads (1~ does n ot
impose this requirement, but instead checks primers of a single length spec-
ified by the user. Both of these approaches eliminate potentially good primers
but will, in most cases, produce an adequate num ber of primers that meet all
of the conditions considered to be important by the investigators.
The researcher using the computer software should keep in mind that the
broader the selection parameters are made, the more cases the comput er must
consider, significantly affecting the time required for primer searches. This
is one reason that search parameters should be kept as narrow and specific
PCR Me t h o d s a n d A p p l i c a ti o n s 3 5
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M an u a l Supp l em en t l l
a s p o s s i b l e w h e n c l e a r l y d i c t a t e d b y e x p e r i m e n t a l d e s i g n . M o r e r e s t r i c t iv e
s e a r c h p a r a m e t e r s u s u a l l y r e s u lt i n fa s t e r s e a r c h e s a n d p r o d u c e p r i m e r s o f
g r e a t e r q u a l i t y . I n p r o g r a m s t h a t a t t e m p t m o r e d i f f i c u l t s e l e c t i o n t a s k s, s u c h
a s c h o o s i n g p r i m e r s t h a t a r e h i g h l y c o n s e r v e d a c r o s s m a n y s p e c ie s , o r s e c t i o n
o f d e g e n e r a t e p r i m e r s f r o m p r o t e i n s e q u e n c e s , t h e b a s i c c ri t e r i a f o r p r i m e r
s e c t i o n o f t e n m u s t b e r e l a x e d b e f o r e t h e s o f t w a r e f i n d s a n y s u i t a b l e p r i m e r
p a i r s .
U s i n g o n e o f t h e a v a i l a b l e s o f t w a r e p r o g r a m s i n c o n j u n c t i o n w i t h t h e
i n f o r m a t i o n p r e s e n t e d h e r e s h o u l d r e s u l t i n t h e s e l e c t i o n o f a g o o d p r i m e r
s e t; t h e n e x t t a s k is t h e p r e p a r a t i o n o f a g o o d n u c l e i c a c i d t e m p l a t e .
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PCR Me t h o d s a n d A p p l i c a t io n s 7
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