general concepts of primer design

8/10/2019 General Concepts of Primer Design

1/9

1993 3: S30-S37Genome Res.C W Dieffenbach, T M Lowe and G S Dveksler

General concepts for PCR primer design.

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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



5/9

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



6/9

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



7/9

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



8/9

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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2. Lowe, T., J. Shareifkin, S.Q. Yang, and C.W. Die ffenba ch. 1990. A com put er p rog ram for

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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


general concepts of primer design

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