Oligo Design for Genetics

8/10/2019 Oligo Design for Genetics

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115

Primer Design(in this case the word Primermeans an oligonucleotide)


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117

Introduction to Primer Design for PCRJayme Olsen

Oligonucleotides, also referred to as primers, are short single strands of nucleic acids that are

synthesized from either DNA or RNA in order to bind to a complementary strand. Primers have

a target area where they bind and act as the starting point for polymerase to extend from, and

thus determine what segment of DNA gets amplified. DNA consists of a double stranded helix.One strand of the DNA is named the sense strand and the other strand is the anti-sense

strand. These two DNA strands are complements of each other. During PCR, the denaturing

step will break the hydrogen bonds, separating the two strands. This allows the primers to anneal

to the target region on the DNA during the annealing step. One primer is designed to anneal tothe sense strand and the other primer needs to bind to the anti-sense strand.

When designing primers for PCR it is necessary to take into consideration things like: how many

primers are needed, the length of the primer, the 5 and 3end, the mutation location in primer,the primer melting/annealing temperature, the G-C content, primer dimmer and the distance

between the forward and reverse primers.

How Many Primers?

When ordering oligonucleotides for your particular CFTR mutation 3 or 4 primers should be

used. Since experiments often fail you cannot design a good PCR diagnostic test wherefailing(a

negative result) is considered a dependable diagnosis. You dont want to tell the parents with ababy who might have CF: We didnt get a band on the gel so she maybe doesnt have CF, or we

just screwed up the gel. Your goal is to design an assay that can diagnose either: (i) if the

mutation *is* present by seeing a band on the gel (ie getting a positive result) or (ii) if the normal

DNA sequence is present you can see a different band on the gel. If you attempt to make only 3primers: The wild-type primer could anneal to the anti-sense strand if the mutation is not present

on the DNA. The mutant primer could be identical to the wild-type primer, annealing to the anti-

sense strand, but with the mutation sequence that will allow it to only anneal if the mutation ispresent in the DNA. The reverse primer could then be the same for both the wild-type andmutant primer. It will anneal downstream in the opposite direction on the sense strand. With

three primers the bands are the same size on the gel, if you use 4 primers you can also design the

experiment so two bands of different lengths/sizes show up on the gel.

Length

The length of the primers need to between 15 and 30 base pairs so that they are long enough for

adequate specificity and short enough for them to anneal to the DNA template.

The 5 and 3end

The primers need to be designed so that the 3 end of the forward primer will extend toward thereverse primer. The 3 end of the reverse primer need to also extend toward the forward primer.The 3 ends of the forward and reverse primers should be facing each other from opposite DNA

strands. This will facilitate the continued replication of the desired strand of DNA. If, for

instance, the 3 ends do not elongate in opposite directions (i.e., toward each other) replication

will not work and a PCR product will not be obtained.


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118

Mutation Location

The best way to distinguish the genotype is to put the mutation on the 3 end of the primer.

Placing the mutation closer to the 5 end of the primer may allow for hairpins to occur, where the

primer skips over the mutant base pair and will re-anneal around it.

Primer Melting Temperature (pretty much the same as the annealing temperature)

The Primer Melting Temperature (Tm) is important for the annealing phase of PCR. Preferred

temperatures should be between 50C and 65C. The forward and reverse primer melting

temperatures should be no more than 2 different. To calculate the Tm see the next page onCalculating Annealing Temperatures.

G-C Content

The G-C content of the primer sequence should be relatively high as it has a direct relationshipwith the Tm. There should be a base composition of G-C of about 50%-60%. The 3 end of the

primer should finish with at least one G or C to promote efficiency in annealing due to the

stronger bonding.

Distance between the Forward and Reverse

The forward primer and the reverse primer should be between 300 and 2,000 base pairs apart.

This distance determines how big the band will be in your gel. Larger bands are easier to see.

If they are too close, the amplified region the product will be too small and run off the gel and ifthey are too big, the product will not make it out of the well. Refer to Ch. 20 in your book.

Beware of Primer Dimer

Primer Dimer is an artifact of PCR where primers bind to each or to themselves other instead of

the template DNA and thus act as their own template to make a small PCR product and appear

faintly on an electrophoresis gel. To avoid primer dimers, be sure there are not manycomplementary areas in the base sequence of your forward and reverse primers where the primerstrands would be able to bind to each other instead of the gene.

Things to Avoid

To avoid non-specific binding, design the primers with high annealing temperatures.

To make sure the primers designed will only bind to the target area submit the sequence

to the BLAST website.

The MgCl2and pH conditions can also be adjusted for improved amplified product.

Watch out for runs of singles bases of Gs, Cs, As, and Ts when developing primers

because they can allow mis-priming.

Keep in mind that the more nucleotide bases that the primer is made up of, the more

expensive they are. The shorter the primers are, the less specificity they have in PCR.


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119

Introduction to Calculating PCR annealing temperatures of oligonucleotide primers

The most crucial factors that need to be optimized in a PCR reaction are the magnesium

concentration, enzyme concentration, DNA concentration and annealing temperature of the

primer. The G+C content of the primers should generally be 40-60% and care should be taken to

avoid sequences that produce internal secondary structures as well as primer dimer whereprimers bind to each other. The annealing temperature for a PCR cycle is generally 3-5 degrees

Celsius below the melting temperature (Tm) of the primer. There are several formulas for

calculating melting temperatures. In all cases these calculations will give you a good starting

point for determining appropriate annealing temperatures for PCR primers. The exact optimumannealing temperature must be determined empirically, however. There are numerous websites

that help with primer design and annealing temperature calculations, search for them. Here's

Promegas website at http://www.promega.com/BioMath.

Basic Melting Temperature Calculations

1) The simplest "rule of thumb" formula is as follows:

Tm=4C x (#Gs + Cs in the primer) + 2C x (# As + Ts).

2) This formula is valid for oligos of less than 14 bases and assumes that the reaction is carriedout in 50mM monovalent cations. For longer primers the formula is modified.

Tm= 64.9C + 41C x (number of Gs and Cs in the primer -16.4)/N

Where N is the length of the primer. For example, Promegas T7 promoter primer

(TAATACGACTCACTATAGGG) is a 20-mer that has 5 Ts, 7 As, 4 Cs, and 4 Gs. Thus, itsmelting temperature would be:

64.9C + 41C x (8-16.4)/20= 47.7 C

3) A third formula calculates the Tm with salt concentrations taken into consideration:

Tm = 81.5 +16.6 (log10[Na+]) + 0.41 (%G+C) 675/n

Where [Na+] is the molar salt concentration ; [K+] = [Na+] and n = number of bases in theoligonucleotide primer.

Other useful formulae are: Nanogram of primer = picomole of primer x 0.325 x # bases

MicroMolar concentration of primer = picomoles of primer/ volume (!L) in which the

primer is dissolved.


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120

ADVANCED APPROACH: Mutation Construction through Site-Directed MutagenesisMitchell Wood

Introduction:

PCR is a powerful tool in molecular biology, specifically for genotypic identification of a

given sample of DNA. Some novel mutations in the CFTR gene have only been noticed in a few

patients; therefore the number of DNA samples with that genotype is limited. Genetic tests thatare generated should cover all mutations known in the CFTR gene, including these novel ones.But to test for these rare mutations positive controls must be found, or generated, to experiment

upon before the test is given to a patient. As an alternative to contacting researchers across the

globe for positive control samples, a relatively simple alternative is to use PCR to replicate DNA

with this rare mutation. This process is called Site-Directed Mutagenesis, which in principle usesimperfect stringency in primer annealing to direct a mutation into the replicated DNA.

Methods:

The length of the primer with the forced mutation is the foremost limitation of thereplicated DNA. When the primer anneals and is replicated with the intentional mismatch, the

resulting PCR product will begin with the 5 end of the primer. Therefore, the length of theprimer used in the allele specific positive control test can not exceed the length of the site-

directed mutagenesis primer. However, the length of the allele-specific primer must not be tooshort (under ~18 base pairs) otherwise it is more probable for non-specific binding on non target

DNA. To minimize the complications that come with a lengthy primer, the forced mutation can

be placed as close to the 3 end of the oligo as possible in order to leave the remaining length to

fit the allele specific primer. Refer to Yaku et al. (2008) for ideas and clarification.

1. Design allele specific primers.

2. To design mutagenic primers, add several nucleotides to the 3 end of your allele specificprimers (the exact number should be determined by Yaku et al (2008)).

3.

Predict the sequence of the PCR product and confirm that it is the one you want.

5-TAC ACG CCC AAG TAC GGT TCCACA-3!Primer with mutation3-CCG TCG ATG TGC GGG TTC ATG CCA AAG TGT CTG-5!DNA Template

Replication with above primer will yield a new DNA template with the forced mutation, but the

DNA segment will only be the length between the forward and reverse primers from above.Therefore the direction of the replication in the next ASPCR will have to be closely watched.

5-TAC ACG CCC AAG TAC GGTTG-3!ASPCR Primer using Yaku Method.

3-ATG TGC GGG TTC ATG CCA ACG TGT CTG-5!Complement to primer with

forced mutation.


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4558 Nucleic

Acids

Research,

Vol.

19,

No. 16

Improved site-directed mutagenesis

method

using

PCR

Oscar

P.Kuipers,

Hein

J.Boot

and

Willem

M.de Vos

Molecular

Genetics

Group

of

the

Department

of

Biophysical

Chemistry,

Netherlands

Institute

for

Dairy

Research

NIZO),

PO Box 20, 6710 BA Ede, The Netherlands

Submitted June

18,

1991

Several

methods for

site-directed

mutagenesis using

PCR have

been

described

in

the last few

years.

One

of the most

rapid,

universal and economical

methods was described

by

Landt

et

al .

1 .

This

procedure

requires just

one

mutagenic primer

and two

universal primers, which may contain convenient

restriction

sites

for cloning.

Essentially,

it

makes

use of two

subsequent

amplification

rounds,

the

first

with

the

mutagenic

oligonucleotide

and th e

antiparallel

universal

primer

and

the second one

using

the

purified first

fragment

as a

primer, together

with th e second

universal primer, and subsequent digestion

and

cloning

of the

fragment. possible problem

described by

these

authors

is

th e

untempl ted

addition of one nucleotide

at

the 3

site-specifically

altered end

of

the

first

amplified

fragment

by Taq-polymerase,

which

can

give rise to

unwanted

mutations in

the second

generated

fragment.

The

authors

advise

to

use lower concentrations

of

dNTPs to

avoid

untemplated

addition of a

nucleotide.

A drawback

of

this

procedure

is

a

lower

yield

and

no

guarantee

for the

absence

of a

3 additional

residue.

A second solution

for the

problem

is

to

remove

the

additional

3 residue

by

the

action of

e.g.

T4-polymerase

prior

to performance

of the second

PCR. This

means that an

additional

enzymatic

modification

step

ha s

to

be

performed,

which

might

not

be

fool-proof.

As has been

observed

by

several authors the 3

additional nucleotide

appears

almost

invariably

to

be an

A-residue

when

using Taq

polymerase

2,

3). Making

use

of

this observation

we have

successfully applied

a modification

in th e method

which can

generally

be used to

exclude the described difficulties. A

mutagenic oligonucleotide

is

used

for the

first PCR reaction

which is

designed

in su h

a

way that the first 5 nucleotide

of

the primer

follows a T-residue

in

the same strand

of

template

sequence. Thus, whether or not

the

amplified

primer

fragment

from

the first

PCR

contains an

additional 3

A-residue,

in both cases

the

amplified product

will

have the

correct

sequence, without need

for further modifications.

Since

in

almost

every

case it should

be possible

to

find a T-residue

at a

reasonable distance

from the site

of

mutation,

this

adjusted

method

is

generally applicable.

We

performed several different

site-directed

mutagenesis

experiments

by this method on the

nisA

gene

4 . The following

experimental conditions

were

used.

Approximately

10

ng of plasmid

DNA harbouring

the nisA

gene

was used

as

template

for PCR in a

total

volume

of 50

Id,

containing

1

of

Taq-polymerase (BRL), 50 mM NaCl, 10 mM

Tris-HCl

pH 8.8,

2 mM

MgCl2, 10

itg

gelatine, 200 zM of

dNTPs,

10

pmol

of

each primer, 2. 5

of stabilizer (1

W-1,

BRL) and covered with 100

,ul

of light

mineral oil. PCR was

performed

in 30

cycles,

each

cycle

consisting of a denaturing

step

at

93C

for

1

min., a primer annealing step at 54C

for

1.5

min. and

an extension

step at

72C

for

2. 5

min. using

a

Bio-

med

Thermocycler 60.

The

DNA-fragments were

purified

by

TAE-agarose

gel

electrophoresis

and

recovered

using the

Gene-

Clean procedure

Bio

101,

La Jolla, California). Fig. 1.

shows

the

sequence

of the nisA

gene and that

of

one

of

the

oligonucleotides we used

for

site-directed

mutagenesis.

In each

case we obtained

the

designed

mutated

fragment

without

undesired

substitutions as was shown

by

dideoxy

sequencing

of

six

independent

clones from each of several

different

mutagenesis

experiments.

This shows

that no

other nucleotide than an

A-

residue,

or

no nucleotide at

all,

had

been

applied

to the 3 end

of

the amplified

primer fragment

although

we

used

up

to 200

AM

of

dNTPs

in

al l

PCR reactions.

In two other

mut genesis

experiments using

the same

PCR

conditions as described

above,

primers were

used which followed another nucleotide than

a

T-

residue

at the

5

end

in

our case a

C-residue). Following

subcloning

of the

digested

fragments,

six clones obtained from

each

mutagenesis

experiment

were

sequenced.

In

ten

out

of

twelve cases a

T for

C substitution was

encountered

on th e

left

side of the

5

end of

the first

mutagenic

primer,

simultaneously

with the desired

mutation. In one case th e

wild-type

sequence

was

observed, probably

originating

from

a cloned

template

fragment and

in

one case the desired

mutation and the

correct

sequence at the

5 end

of the

primer

were

found.

Thus,

when

using

this

mutagenesis method,

a

well considered choice

of

primer

sequences

can

considerably

increase th e

frequency

of

correctly

mutated

sequences.

REFERENCES

1.

Landt,O., Grunert,H.-P.

and

Hahn,U.

1990)

Gene

96

125-128.

2.

Clark,J.M.

1988) Nucl.

Acids

Res.

16,

9677-9686.

3. Mole,S.E., Iggo,R.D. and Lane,D.P.

1989)

Nucl.

Acids

Res.

17,

3319.

4.

Buchman,G.W., Banerjee,S.

and Hansen,J.N.

1988)

J. Biol.

Chem. 263,

16260-16266.

5

T AOGTTAG 3

PstI)

S5*ACAAGTAr2TCZCTA-TGACACCCGG2TG

3

CATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTCTGATGGGTTGTAACATGAAAACAGCAA

CTTGTCATTGTAGTATTCACGTAAGCAAATAACCAAATCAAAGGATAGTATTTTGTTAGTTCAGACATGGATAC

TATCCTATTTTTATAAGTTATTTAGGG

3

3

GGATAAAAATATTCQAAAAATCCC

5

(HindIII)

Figure

1.

Sequence

of nisA and

primers

used for

PCR. Primers ar e shown in

bold,

the

template T-residue

5

to the

mutagenic

primer Ser5

Ala)

is indicated

by

an

asterisk and sites

of

mutation ar e

underlined.

The

non-coding sequence

of

the nisA

gene

is

shown in italics.

l. 1991

Oxford

University

Press


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122

ADVANCED APPROACH: Introduction to The Yaku-Bonczyk Primer Design MethodVincent Cracolici

The What?

The Yaku-Bonczyk method is an advanced protocol by which primers can be designed in

order to increase PCR stringency as well as decrease the chance of false positives or negatives

seen in a gel. In 2008, Yaku et al presented this design method; it was then further investigatedby LB 145 student Sarah Bonczyk in spring 2009. By slightly altering the classic 3 singlebase pair difference between wild-type and mutant primers, a research team can drastically

increase primer discrimination against nonspecific binding. Similar results were shown by

Wittwer et al (1993).

How does it work?

The standard method of primer design for a genetic mutation, like one on CFTR,

typically involves two forward primers which are identical save for the base pair nearest the 3

end: one primer is complementary to wild-type DNA and the other to mutant DNA. However,the single base-pair mismatch between these two primers is often not enough to ensure that the

wild-type primer will not anneal to and extend mutant DNA, and vice versa.The Yaku-Bonczyk method differs from the standard because the primers are designed to

better discriminate against non-complementary DNA by always incorporating an intentionalmismatch into the primer. The Yaku-Bonczyk method involves the most 3 base pair of each

forward primer again being complementary to the mutant/wild-type DNA it is seeking, the

second base pairs in are designed to always anneal to either type of DNA, and the third base

pairs in are designed as an intentional mismatch that will never anneal to either type of DNA (seeillustration).

If a primer and its complementary DNA strand anneal to each other, the single mismatch

three base pairs in from the 3 end is not enough to prevent extension and will result in only asmall hairpin in the sequence. Additionally, if a primer and a non-target DNA strand anneal to

each other, the complementary match at the second base pair from the 3 end of the primer is notstrong enough to pull the two strands together and is unlikely to allow for extension. Therefore,

nonspecific binding is decreased.

Points to Ponder

-The intentional mismatch that exists on all the primers three base pairs in from the 3 end

provides an excellent opportunity to boost your primers G/C content.-Should this intentional mismatch still be included in the calculations of annealing temperatures?

-Remember to consider purine/pyrimidine interactions with themselves and each other in

designing the intentional mismatch.


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123

The Yaku-Bonczyk Method

Primer: 5 AACGTGGTCXYZ 3

X: Should be designed to NEVERanneal to mutant ORwild-type DNAY: Should be designed to ALWAYSanneal to mutant ANDwild-type DNA

Z: Should be site specific: anneal to EITHERmutant OR wild-type DNA

A primer designed with the Yaku-Bonczyk method will anneal to and extend target DNA despite

the intentional mismatch. The force of the single repulsion will not hinder the primer as a whole.

A primer designed with the Yaku-Bonczyk method will not anneal to nor extend non-target DNAas a result of the two mismatches. The attraction at the second base pair in on the primer is not

enough to allow for extension at the 3 end.


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Research Article

Design of allele-specific primers anddetection of the human ABO genotypingto avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambdaDNA at the 30 end were carried out in order to develop allele-specific primers capable of

detecting SNP in genomes without generating pseudopositive amplification products, and

thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products

were obtained when primers forming a single or two mismatch pairings at the 30 end were

used. In particular, 30 terminal A/C or T/C (primer/template) mismatches tended to allow

PCR amplification to proceed, resulting in pseudopositive results in many cases. While lessPCR product was observed for primers forming three terminal mismatch pairings, target

DNA sequences were efficiently amplified by primers forming two mismatch pairings next

to the terminal G/C base pairing. These results indicate that selecting a primer having a 30

terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that

form mismatch pairings with the template sequence can be used as an allele-specific primer

that eliminates the pseudopositive problem. Trials with the human ABO genes demon-strated that this primer design is also useful for detecting a single base pair difference in

gene sequences with a signal-to-noise ratio of at least 45.

Keywords:

Allele-specific primer / Human ABO gene / Pseudopositive problem / SNP30 terminal mismatch pairings DOI 10.1002/elps.200800097

1 Introduction

Among gene polymorphisms, SNP occur at the highestfrequency. SNP are reported to occur at a frequency of about

0.1% in the human genome, and more than three million

SNP have been identified [1]. Research on SNP in humans

has revealed several associations of SNP types with diseasesincluding diabetes, cancer, and myocardial infarction, and

SNP in the human genome are also known to influence

aspects of the human constitution such as blood group type

and the sensitivity to alcohol [27].

Several techniques for SNP genotyping have been

reported. These include utilizing DNA hybridization [8],primer extension reaction using allele-specific DNA primers

and DNA polymerase [912], DNA mismatch-recognizing

enzymes [1315], the Invader assay [16, 17], DNA chips [18,19], and pyrosequencing [2022]. Among these, the methodusing allele-specific primers has been investigated exten-

sively for its advantages in cost, reaction time, and simplicity

of handling. Allele-specific DNA primers exhibit different

efficiencies for primer extension reactions, depending on

the identities of the base pairs of the SNP in the template

DNA, and the SNP genotyping can be achieved simply by

detecting the amounts of PCR products or even by detectingthe pyrophosphate generated during PCR [9, 10].

Proper design of primer DNA sequences is important for

the efficient detection of SNP by PCR. The allele-specificprimers are usually designed to complement template DNA

and contain a nucleotide specific to the SNP at the 30 end. The

SNP-specific nucleotide forms a base pairing or mismatch

pairing depending on the base pair identity of the SNP and

only proper base pairing at the end of the primer/template

duplex is effective in producing PCR products, while less PCR

product is produced for terminal mismatch pairings due todecreased DNA polymerase binding and inefficiencies in

incorporating 20-deoxyribonucleoside triphosphates [23].

Nevertheless, unexpected primer extension with mismatch-forming DNA primers, the so-called pseudopositive problem,may occur when PCR is carried out under unsuitable reaction

conditions with regard to the amplification cycle, reaction time,

temperature, and 20-deoxyribonucleoside triphosphates

concentration, although each of these conditions may be

optimized through repeated trials. The pseudopositive

problem also arises due to specific DNA primer sequences.Primer extension reactions are often observed when a single

Hidenobu Yaku1,2

Tetsuo Yukimasa1

Shu-ichi Nakano2

Naoki Sugimoto2,3

Hiroaki Oka1

1Advanced Technology Research

Laboratories, Matsushita ElectricIndustrial Co. Ltd., Kyoto, Japan2Frontier Institute for

Biomolecular EngineeringResearch, Konan University,Kobe, Japan3Department of Chemistry,

Faculty of Science andEngineering, Konan University,Kobe, Japan

Received February 7, 2008

Revised May 14, 2008

Accepted May 21, 2008

Additional corresponding author: Dr. Hiroaki Oka,

E-mail: [email protected]

Correspondence: Hidenobu Yaku, Advanced Technology

Research Laboratories, Matsushita Electric Industrial Co. Ltd.,

3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto, Japan

E-mail:[email protected]

Fax: 181-774-98-2585

Electrophoresis2008, 29, 413041404130


10/19

mismatch is formed at the 30 end with the primer used

[2325]. The pseudopositive problem becomes much more

serious for the allele frequency analysis than when an SNP

typing is aimed at distinguishing homozygotes and hetero-

zygotes because the pseudopositive signals should be less than1% of those obtained with matched primer in the case of the

allele frequency analysis. So, several strategies have been

explored to eliminate the pseudopositive problem. Kambara

and coworkers [9, 10] designed allele-specific primers so that

the 30 end nucleotide was specific to the SNP and the 3rd

nucleotide from the 30 end was mismatched with the template

DNA. Aonoet al.[12] developed allele-specific primers so that

the 2nd nucleotide from the 30 end was specific to the SNP and

the 3rd nucleotide from the 30 end was mismatched with thetemplate DNA. In addition, several methods using modified

primer, such as locked nucleic acid primer [13], phosphor-

othioate-modified primer [11, 14], and dideoxynucleotide-

terminated primer [15] have been developed. Zhanget al.[11]

Table 1. Sequences of lambda DNA and the forward primers used for PCR

a) Underlined nucleotides in the forward primers are unpaired with the lambda DNA sequence.

Electrophoresis2008, 29, 41304140 Nucleic acids 4131

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com


11/19

reported a method using an allele-specific primer in which the

30 end nucleotide was a phosphorothioate-modified nucleotideand specific to the SNP along with use of a DNA polymerase

with proofreading function. Among these studies, allele-

specific DNA primers forming mismatch pairings near the

SNP distinction site were used in order to inhibit DNA poly-

merase association only when forming the mismatch pairingat the SNP site and to eliminate pseudopositive PCR products.However, the appropriate values of the number, position, and

type of the mismatch nucleotide pairs have not yet been

systematically examined.

To investigate whether the use of allele-specific primersequences can eliminate the pseudopositive problem, we

carried out systematic PCR experiments using lambda DNA

as a template and DNA primers designed to form different

numbers and different types of mismatch pairings near the30 end. DNA primers designed to form three consecutive

mismatch pairings at the 30 end produced less PCR product,even after 30 amplification cycles, while DNA primers

forming two mismatch pairings next to the G/CWatson-Crick base pairing produced moderate amounts of

PCR product. These results demonstrate that primers forwhich the 30 nucleotide specific to the SNP and the other

two nucleotides were mismatched with the template DNA

could amplify specific alleles and would be useful for SNP

genotyping. Moreover, primer design was applied to thedetection of human blood types, and properly designed

primers were shown to allow efficient detection of single

base pair differences in the ABO gene without the pseudo-

positive problem.

2 Materials and methods

2.1 PCR using lambda DNA

Sequences of lambda DNA (TAKARA BIO) that was used asa template DNA, and 41 synthetic oligoDNAs (Proligo,

E@sy OligosTM) that were used as DNA forward and reverse

primers are presented in Table 1. Forward primer nos. 140

(50-GATGAGTTCGTGTCCGTACAACX3X2X1-30) are

complementary to base pairs 71317155 of the lambda

DNA sequence, forming zero, one, two, or three mismatchpairings at the 30 end depending on the identity of X1, X2,

and X3. The reverse primer sequence (50-GAATCACGG-

TATCCGGCTGCGCTGA-30

) was fully matched with basepairs 74067430 of the lambda DNA (see Table 1).

After initial denaturation at 951C for 10 min, the

amplification was carried out for 20 or 30 cycles as follows in

a LightCycler (Roche Diagnostics) thermal cycler: dena-

turation at 951C for 10 s, annealing at 581C for 10 s, and

DNA extension at 721C for 10 s. The 20-mL PCR mixtures

were prepared using the LightCycler FastStart DNA MasterSYBR Green I reaction kit (Roche Diagnostics, with 1 ng/mL

lambda DNA, 1.25 mM MgCl2, 1 mM forward primer, and

1 mM reverse primer. PCR products were analyzed by elec-

trophoreses on 3% agarose gel on a Mupid (ADVANCE Co.)

followed by the quantification of the PCR products by

Agilent 2100 Bioanalyzer (Agilent Technologies).

2.2 PCR of the human ABO blood type genes

The 22nd base pair in exon 6 of the ABO gene was selectedas a target for human blood genotyping. As given in Table 2,this base pair is a G/C base pair in the A and B alleles and

an A/T base pair in the O allele due to deletion of the G/C

base pair found in the A and B alleles [3, 4]. Accordingly, the

22nd base pair in exon 6 is the homo G/C base pair forblood type AB and the homo A/T base pair for blood type O.

Two-step PCR was carried out to detect allelic difference

in the 22nd base pair of exon 6 of the ABO gene. In the 1st

PCR, a fragment of exon 6 in human genomic DNA wasamplified by using a forward primer (50-TAGGAAG-

GATGTCCTCG-30) complementary to base pairs 117 and areverse primer (50-TTCTTGATGGCAAACACAGTTAAC-30)

(Proligo, E@sy OligosTM

) complementary to base pairs112135 of the A and B alleles or base pairs 111134 of the

O allele on exon 6. The 1st PCR was carried out in 20 mLreactions using the LightCycler FastStart DNA Master SYBR

Green I reaction kit with 0.5 ng/mL of genomic DNA,

1.25 mM MgCl2, and 1 mM of each of the forward and

reverse primers. Following denaturation at 951C for 10 min,50 cycles of denaturation at 951C for 10 s, annealing at 521C

for 10 s, and extension at 721C for 10 s were carried out on

the LightCycler. Amplification was monitored in real time

by measuring the fluorescent intensity of SYBR Green I,

and amplifications were confirmed to be completed by the

50th cycle with the amount of amplification product being

almost identical for both alleles (data not shown).In order to analyze ABO genotyping with allele-specific

primers, the 2nd PCR was carried out using the product of the

1st PCR as a template and the allele-specific forward primersgiven in Table 3 (50-TAGGAAGGATGTCCTCGTGY3Y2G-3

0).The 30 end nucleotide of the primers is G, which is comple-

mentary to the C of the 22nd G/C base pair of exon 6 in the A

Table 2. Sequences of exon 6 of the ABO gene

ABO

gene

Sequence (5-3)a)

A allele

andB allele

1 TAGGAAGGAT GTCCTCGTGG TGACCCCTTG GCTGGCTCCC

41 ATTGTCTGGG AGGGCACATT CAACATCGAC ATCCTCAACG

81 AGCAGTTCAG GCTCCAGAAC ACCACCATTG GGTTAACTGT

121 GTTTGCCATC AAGAA

O allele 1 TAGGAAGGAT GTCCTCGTGG TACCCCTTGG CTGGCTCCCA

41 TTGTCTGGGA GGGCACATTC AACATCGACA TCCTCAACGA

81 GCAGTTCAGG CTCCAGAACA CCACCATTGG GTTAACTGTG

121 TTTGCCATCA AGAA

a) Underlined nucleotides, the G and the A, are the 22nd

nucleotide in the AB allele and the O allele, respectively.

Electrophoresis2008, 29, 413041404132 H. Yaku et al.



12/19

and B alleles but is mismatched with the A/T base pair at base

pair 22 of the O allele. The 2nd and 3rd nucleotides from the30 end, Y2and Y3, respectively, are designed to be mismatched

with the 20th and 21st nucleotides of the exon 6 sequence.

The reverse primer used for the 2nd PCR was 50-TTCT

TGATGGCAAACACAGTTAACC-30. The PCR mixtures

(20mL) contained 2mL of the 1st PCR product diluted 1000-times, 1.25 mM MgCl2, 1 mM of each of the forward andreverse primers. Following DNA denaturation at 951C for

10 min, the 2nd PCR was carried out for 2128 cycles of

denaturation at 951C for 10 s, annealing at 521C for 10 s, and

extension at 721C for 10 s. Experiments using lambda DNAand exon 6 of the human ABO gene were highly reproducible,

and the concentrations of the PCR product presented here are

averages of three independent experiments.

3 Results and discussion

3.1 DNA primer design for PCR of lambda DNA

The allele-specific primers are expected to result insuccessful PCR amplification only when the nucleotide

specific to the SNP is present in the primer and is

complementary to the SNP nucleotide of the analyte DNA.

However, allele-specific primers with SNP-specific nucleo-tides at the 30 end and the other bases of the primer, which

are fully complementary to the template sometimes, result

in amplification, even when the SNP-specific nucleotide is

not complementary to the SNP nucleotide. It has been

reported that the PCR products can be suppressed by usingmismatch-forming forward primers that form mismatchpairs in addition to the SNP distinction nucleotide [9, 10,

12]. To obtain information on PCR primer design, primers

with different mismatches and PCR cycle numbers were

tested with the lambda DNA template. As shown in Fig. 1and Table 1, PCR experiments were primarily carried out

with 13 forward primers (nos. 113, 50-GATGAGTTCGT

GTCCGTACAACX3X2X1-30) that were complementary to

the 7131st7155th base pairs of the lambda DNA: primerno. 1 has a sequence that is fully complementary with the

template DNA (50-TGG-30/50-CCA-30, where 50-TGG-30 isX3X2X1 at the 3

0 end of the primer and 50-CCA-30 is the

complementary sequence of the lambda DNA), primer nos.24 have a single mismatch at their 3 0 end (50-TGX1-3

0/50-

CCA-30, where X15A, T, or C forming mismatch pairingsof A/C, T/C, or C/C, respectively), and primer nos. 513

have mismatches on the two terminal base pairs (50-TX2X1-

30/50-CCA-30, where X2X15CA, CT, CC, AA, AT, AC, TA,

TT, or TC). These primer sequences cover all possiblemismatch pairings with the template DNA sequence.

3.2 PCR using mismatch-forming primers with the

lambda DNA

PCR products using the lambda DNA as a template wereanalyzed for electrophoretic mobility. Amplification

products were expected to be 300 bp in accordance with

the forward and the reverse primer binding sites (seeTable 1). However, electrophoretic assay of amplification

products produced with primer no. 1 showed a single

product of about 270 bp in length, based on comparisons to

marker DNA fragments (data not shown). The substitution

of dUTP for dTTP in the PCR reaction kit was confirmed to

Taq polymeraseVariant

nucleotidesForward primers

5-GATGAGTTCGTGTCCGTACAACX3X2X13

Lambda DNA5. . .GATGAGTTCGTGTCCGTACAACTGG. . . . .

3. . .CTACTCAAGCACAGGCATGTTGACC. . . . .

7131

. . . . .AGTCGCGTCGGCCTATGGCACTAAG. . .5

. . . . .TCAGCGCAGCCGGATACCGTGATTC. . .3

7155

7406 7430

3-AGTCGCGTCGGCCTATGGCACTAAG-5

Reverse primer

Comparison of the concentrations of the PCR products

Figure 1. Schematic diagram of the PCR

experiments using lambda DNA and the

forward primers forming zero, one, or two

mismatch nucleotide pairings.

Table 3. The allele-specific forward primers used for the

detection of single base pair difference in the AB

allele and the O allele

Allele specific primer Sequence (5-3)a)

ABO261 AAG TAGGAAGGATGTCCTCGTGAAG

ABO261 ACG TAGGAAGGATGTCCTCGTGACG

ABO261 AGG TAGGAAGGATGTCCTCGTGAGGABO261 CAG TAGGAAGGATGTCCTCGTGCAG

ABO261 CCG TAGGAAGGATGTCCTCGTGCCG

ABO261 CGG TAGGAAGGATGTCCTCGTGCGG

ABO261 TAG TAGGAAGGATGTCCTCGTGTAG

ABO261 TCG TAGGAAGGATGTCCTCGTGTCG

ABO261 TGG TAGGAAGGATGTCCTCGTGTGG

a) Underlined nucleotides are unpaired with the AB allele

and the O allele.




13/19

considerably affect mobility of the amplified fragments inthe polyacrylamide gel, and products amplified with the

primer no. 1 corresponded to a length of about 300 bp if

thymine was incorporated compared with a length of

270 bp. Only a single amplification product was observed

in the experiments, which were highly reproducible.

Thus, we concluded that the PCR product with a lengthof 270 bp based on marker DNAs was the target PCR

product.

The effect of PCR cycle number on SNP detection andthe specificity of allele-specific primers was examined for 20and 30 cycles. PCR with a single mismatch at the 3 0 end

between primers (nos. 24) and templates produced

moderate amounts of product about 300 bp in length after

20 (Fig. 2A) and 30 cycles (Fig. 2B). The fully complemen-

tary primer (no. 1) produced PCR products of 260 and

390 nM after the 20 and 30 cycles, respectively, suggestingthat the amplification reaches a plateau by the 20th cycle.

When primer nos. 2 and 3 forming a single terminal A/C or

T/C mismatch pairing, respectively, were used, the amounts

of the PCR product obtained after the 20th cycle (about

200 nM) were more than 70% of those obtained by the fullycomplementary primer, and the amounts after 30 cycles

with primers nos. 13 were almost identical (about 400 nM).

Primer no. 4 formed a single C/C mismatch pairing at the

end, which inhibited amplification after 20 cycles (less than

50 nM). These results are in agreement with those of Huang

et al. [23] and Kwok et al. [24], demonstrating much lowerprimer extension efficiency with terminal C/C mismatch

compared with A/C and T/C mismatches. The thermo-

stability of the primertemplate duplex may not affect theefficiency based on a thermodynamics study by Allawi et al.[29] showing that the T/C mismatch, as well as the C/C

mismatch, destabilized the DNA duplex. On the other hand,

even primer no. 4 showed additional amplification after 30

cycles. The terminal C/C, A/G, and G/A mismatches impair

amplification efficiency compared with complementarypairing [23]. However, based on the results shown in

Figs. 2A and B, cycle number is shown to strongly affect the

concentration of amplification products. Consequently, only

a single terminal mismatch is thought to be insufficient foreliminating the pseudopositive problem.

0

50

100

150

200

250

300A

B

1 2 3 4 5 6 7 8 9 10 11 12

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3AC

C5

5TC

A3

3AC

C5

5TC

T3

3AC

C5

5TC

C3

3AC

C5

5TA

A3

3AC

C5

5TA

T3

3AC

C5

5TA

C3

3AC

C5

5TT

A3

3AC

C5

5TT

T3

3AC

C5

5TT

C3

3AC

C5

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3AC

C5

5TC

A3

3AC

C5

5TC

T3

3AC

C5

5TC

C3

3AC

C5

5TA

A3

3AC

C5

5TA

T3

3AC

C5

5TA

C3

3AC

C5

5TT

A3

3AC

C5

5TT

T3

3AC

C5

5TT

C3

3AC

C5

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3AC

C5

5TC

A3

3AC

C5

5TC

T3

3AC

C5

5TC

C3

3AC

C5

5TA

A3

3AC

C5

5TA

T3

3AC

C5

5TA

C3

3AC

C5

5TT

A3

3AC

C5

5TT

T3

3AC

C5

5TT

C3

3AC

C5

Singlemismatch

Twomismatches

Conce

ntration

ofamplificatio

nproducts(nM)

Concentration

ofamplificationproduct

s(nM)

Fullymatched

Fully

matched

0

50

100

150

200

250

300350

400

450

1 2 3 4 5 6 7 8 9 10 11 12

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3ACC5

5TC

A3

3ACC5

5TC

T3

3ACC5

5TC

C3

3ACC5

5TA

A3

3ACC5

5TA

T3

3ACC5

5TA

C3

3ACC5

5TT

A3

3ACC5

5TT

T3

3ACC5

5TT

C3

3ACC5

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3ACC5

5TC

A3

3ACC5

5TC

T3

3ACC5

5TC

C3

3ACC5

5TA

A3

3ACC5

5TA

T3

3ACC5

5TA

C3

3ACC5

5TT

A3

3ACC5

5TT

T3

3ACC5

5TT

C3

3ACC5

5TG

G3

3ACC5

5TG

A3

3AC

C5

5TG

T3

3AC

C5

5TG

C3

3ACC5

5TC

A3

3ACC5

5TC

T3

3ACC5

5TC

C3

3ACC5

5TA

A3

3ACC5

5TA

T3

3ACC5

5TA

C3

3ACC5

5TT

A3

3ACC5

5TT

T3

3ACC5

5TT

C3

3ACC5

Singlemismatch

Twomismatches

13

13

Figure 2. Concentrations of

the target PCR products after

20 (A) or 30 (B) amplification

cycles using lambda DNA

and the forward primers

forming zero, one, or two

mismatch nucleotide pair-

ings.




14/19

In contrast with primers producing single mismatches,

less PCR product was produced after 20 cycles with the

primer nos. 513, which have two terminal mismatch pairs(Fig. 2A). In particular, primers forming the mismatched

pairings, C/C, A/C, or T/C, next to the C/C mismatched

pairing at the end (nos. 7, 10, and 13, respectively) showedinhibited amplification, even after 30 cycles. Because primerno. 4 that formed a G/C pair next to a C/C mismatch pair at

the end resulted in amplification product, the genotyping of

G and C in analyte DNA can be accomplished by using these

primers. On the other hand, moderate amounts of amplifi-

cation product after 30 cycles (230260 nM) were obtainedfor reactions with primers forming A/C or T/C mismatches

at the end (nos. 6, 8, 9, 11, and 12) but not for reactions with

primer no. 5. Instead of the 300-bp product produced with

other primers, the product produced with primer no. 5 waslonger due to primer hybridization at an unexpected site.

These results suggest that the amplification product ofreactions with primers with two mismatches depends on the

PCR cycle number, the identities of the mismatches, and

that the terminal A/C and T/C mismatch pairs have lessinfluence on PCR efficiency. Moreover, primers forming

A/C or T/C terminal pairing produced similar amounts of

amplification product after 30 cycles, indicating that the

penultimate mismatch has much less effect on PCR effi-

ciency than the 30end mismatch type of primers with two

consecutive mismatches at the end. Consequently, primers

forming one or two mismatches at the 30 end, with theexception of primers forming terminal C/C mismatch

pairings, proved to be unsuitable for avoiding pseudoposi-

tive amplifications.

3.3 PCR using the primers with three mismatched

nucleotides at 30 end

Results of the previous section suggest that terminal A/C or

T/C mismatch pairing allows amplification after 30 cycleswith primers forming two mismatches at the end and

potentially leading to the pseudopositive problem for SNP

detection. Thus, we examined PCR using the primer nos.

1431, which had 30 terminal A/C or T/C mismatch pairing

0

50

100

150

200

250

300A

B

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

5ACA

3

3ACC

55A

CT3

3AC

C55A

AA3

3AC

C5

5AAT

3

3ACC

55A

TA3

3ACC

5

5ATT

3

3ACC

55G

CA3

3ACC

55G

CT3

3AC

C55G

AA3

3AC

C55G

AT3

3AC

C55G

TA3

3AC

C55G

TT3

3AC

C55C

CA3

3AC

C55C

CT3

3AC

C5

5CA

A3

3AC

C55C

AT3

3AC

C55C

TA3

3AC

C55C

TT3

3AC

C5

24

5ACA

3

3ACC

55A

CT3

3AC

C55A

AA3

3AC

C5

5AAT

3

3ACC

55A

TA3

3ACC

5

5ATT

3

3ACC

55G

CA3

3ACC

55G

CT3

3AC

C55G

AA3

3AC

C55G

AT3

3AC

C55G

TA3

3AC

C55G

TT3

3AC

C55C

CA3

3AC

C55C

CT3

3AC

C5

5CA

A3

3AC

C55C

AT3

3AC

C55C

TA3

3AC

C55C

TT3

3AC

C5

5ACA

3

3ACC

55A

CT3

3AC

C55A

AA3

3AC

C5

5AAT

3

3ACC

55A

TA3

3ACC

5

5ATT

3

3ACC

55G

CA3

3ACC

55G

CT3

3AC

C55G

AA3

3AC

C55G

AT3

3AC

C55G

TA3

3AC

C55G

TT3

3AC

C55C

CA3

3AC

C55C

CT3

3AC

C5

5CA

A3

3AC

C55C

AT3

3AC

C55C

TA3

3AC

C55C

TT3

3AC

C5

Threemismatches

Threemismatches

Con

centration

ofamplifica

tionproducts(nM)

0

50

100

150

200

250

300

350

400

450

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

5ACA

3

3AC

C55A

CT3

3ACC

5

5AA

A3

3ACC

5

5AAT3

3ACC

55AT

A3

3ACC

55A

TT3

3ACC

5

5GCA

3

3AC

C5

5GCT

3

3ACC

5

5GAA

3

3ACC

5

5GAT

3

3ACC

5

5GTA

3

3ACC

5

5GTT

3

3ACC

5

5CC

A3

3ACC

5

5CC

T3

3ACC

5

5CAA

3

3ACC

5

5CA

T3

3ACC

5

5CTA

3

3AC

C5

5CT

T3

3ACC

5

5ACA

3

3AC

C55A

CT3

3ACC

5

5AA

A3

3ACC

5

5AAT3

3ACC

55AT

A3

3ACC

55A

TT3

3ACC

5

5GCA

3

3AC

C5

5GCT

3

3ACC

5

5GAA

3

3ACC

5

5GAT

3

3ACC

5

5GTA

3

3ACC

5

5GTT

3

3ACC

5

5CC

A3

3ACC

5

5CC

T3

3ACC

5

5CAA

3

3ACC

5

5CA

T3

3ACC

5

5CTA

3

3AC

C5

5CT

T3

3ACC

5

5ACA

3

3AC

C55A

CT3

3ACC

5

5AA

A3

3ACC

5

5AAT3

3ACC

55AT

A3

3ACC

55A

TT3

3ACC

5

5GCA

3

3AC

C5

5GCT

3

3ACC

5

5GAA

3

3ACC

5

5GAT

3

3ACC

5

5GTA

3

3ACC

5

5GTT

3

3ACC

5

5CC

A3

3ACC

5

5CC

T3

3ACC

5

5CAA

3

3ACC

5

5CA

T3

3ACC

5

5CTA

3

3AC

C5

5CT

T3

3ACC

5

Threemismatches

Threemismatches

Concentra

tion

ofamplificationpro

ducts(nM)

Figure 3. Concentrations of

the target PCR products

after 20 (A) or 30 (B) ampli-

fication cycles using lamb-

da DNA and the forward

primers with 30 terminal

A/C or T/C mismatch pairing

and mismatch pairing in the

adjacent two nucleotides.

G A T GA G T T C G T G T C C G T A C A A CT

C T A C T C A A G C A C A GG C A T G T T G A C C

AC5

3

3

5

Figure 4. Schematic diagram of the hybridization of primer no.

15 and template.




15/19

with the template along with mismatches at the adjacenttwo positions (50-X3X2X1-3

0/50-CCA-30, where X15A or T,and X3X25AC, AA, AT, GC, GA, GT, CC, CA, or CT). The

amount of amplification product expected by using these

primers, with the exception of primer no. 15, was small after

20 cycles (Fig. 3A) and even by the 30 cycles (Fig. 3B). These

observations suggest that primers forming three mismatch

pairings at the 30 end result in less amplification product,regardless of mismatch type and number of PCR cycles.

Primer no. 15 unexpectedly resulted in PCR product after

the 30 cycles due to hybridization at the target binding site

by forming a bulge structure that avoided the three terminal

mismatches (Fig. 4) and, thus, the base pairs near the end

no longer inhibited the amplification reaction.

3.4 Design of DNA primers with two mismatch pairs

adjacent to the terminal base pair

Three types of DNA primer designs for forming three

mismatches at the end were considered for the SNP

detection (Fig. 5): Type 1, the 30 end nucleotide is specific

to the SNP and the next two nucleotides are mismatchedpairings; Type 2, the 2nd nucleotide from the 30 end is

specific to the SNP and the adjacent nucleotides on eitherside are mismatch pairings; Type 3, the 3rd nucleotide from

the 30 end is specific to the SNP and the other nucleotides

form mismatch pairings. Clarification of the amount of the

PCR product produced when S0

is paired with S and thereduced amount of product when S0 is unpaired with S for

the SNP detections (Fig. 5) is required. In fact, Kambara and

coworkers [9, 10] have already reported reduced efficiency of

primer extension for primer DNA corresponding to type 2design even when S0 is paired with S. Additionally, although

type 3 primers form two mismatches, the S0 pairing with S

was found to produce amplification product depending on

the mismatch and PCR cycles, as shown in Figs. 2 and 3.

Therefore, we further investigated DNA primers of type 1

design.To examine type 1 primers, PCR experiments using the

primer nos. 3240 (Table 1) were tested. Primer nos. 3240

were designed to form two mismatched base pairs adjacentto the terminal G/C pair when associating with the lambdaDNA template (50-X3X2G-3

0/50-CCA-30, where X3X25AC,

AA, AT, GC, GA, GT, CC, CA, or CT). The terminal G/C

pair was supposed to distinguish the SNP nucleotide based

of the type 1 primer design. As a result, substantial amounts

of the product (150320 nM) were obtained by all primer

nos. 3240 after 30 cycles (Fig. 6) and six of the primersprovided PCR product of more than 50 nM, even after 20

cycles (data not shown). The results indicate that the type 1

primers are promising as allele-specific primers without the

pseudopositive problem. Importantly, less target PCR

SS

XY

X Y S

XYS

XYS

X Y

SS

X

X

Y

Y

SS

X

X Y

Y

YX S

Y

SXY

S X

Type-1

Type-2

Type-3

Sis paired with S

primer

template

35

53

53

53

53

53

53

primer

templateprimer

template

primer

template

3

5

3

5

primer

template

primer

template

3

5

3

5

3

5

Sis unpaired with S

SSX YSSX Y SX Y SX Y

SSX YSSX Y

SS X YSS X Y

YX S YX S

YS X YS X

3

3

5

5

Figure 5. Candidates for the

allele-specific primers. S and

S0 indicate SNP in the

template DNA and the corre-

sponding SNP nucleotide in

the primer, respectively.

X/X and Y/Y are mismatch

pairings.

0

20

40

60

80

100

120

140

160A

B

32 33 34 35 36 37 38 39 40

32 33 34 35 36 37 38 39 40

5ACG

3

3ACC

55A

AG3

3ACC

55A

TG3

3ACC

55G

CG3

3ACC

55G

AG3

3ACC

55G

TG3

3ACC

55C

CG3

3ACC

55C

AG3

3ACC

55C

TG3

3ACC

5

5ACG

3

3ACC

55A

AG3

3ACC

55A

TG3

3ACC

55G

CG3

3ACC

55G

AG3

3ACC

55G

TG3

3ACC

55C

CG3

3ACC

55C

AG3

3ACC

55C

TG3

3ACC

5

Concentration

ofamplificationpro

ducts(nM)

0

50

100

150

200

250

300

350

Conc

entration

ofamplificationproducts(nM)

Figure 6. Concentrations of the target PCR products after 20 (A)

or 30 (B) amplification cycles using lambda DNA and forward

primers designed to have 30 terminal G/C base pairing and two

mismatched base pairings adjacent to the G/C pair.




16/19

product was observed for DNA primers with three conse-

cutive mismatches, even after the 30 amplification cycles

(Fig. 3B), suggesting that the type 1 primers can prevent the

pseudopositive problem independent of the number of PCR

cycles. These reaction conditions allow SNP genotyping with

DNA template of an unknown concentration.Comparison of amplification data for the primers with

two mismatches (Fig. 2) and three mismatches (Fig. 3) also

give insights into the type 3 primer design in that the 3rd

nucleotide from the 30 end is specific to the SNP. Primers

with two mismatches, including the terminal T/C or A/C

pairing produced PCR products by 30 amplification cycles,

while less product was formed by the primers with three

mismatches. However, the signal-to-noise ratios were smaller

than for the type 1 primers and all primers forming two

mismatches produced less target PCR product (o50 nM)after 20 amplification cycles, indicating that SNP genotyping

of the type 3 primer depends on the number of amplification

cycles. Therefore, the DNA primer design based on the type 1

detection is more promising than that of type 3.

Exon 6 of ABO gene

Isolated genomic DNA

from AB or O type blood

5

3

3

5

5-TTCTTGATGGCAAACACAGTTAAC-3

5-TAGGAAGGATGTCCTCG-3

1st PCR

53

35

5-TTCTTGATGGCAAACACAGTTAAC-32nd PCR

Electrophoresis

Y2 and Y3 are mismatched with the template DNA sequence

[Y3,Y2]=[AA],[CA],[TA],[AC],[CC],[TC],[AG],[CG] or [TG]

5-TAGGAAGGATGTCCTCGTGY3Y2G-3

ABO261AAG, CAG, TAG, ACG, CCG, TCG, AGG, CGG or TGG

Figure 7. Schematic diagram for the detection

of single base pair difference in exon 6 of the

ABO gene using allele-specific primers.

M AB

1O M AB

2O M AB

3O M AB

4O M AB

5O M AB

6O

M AB

7O M AB

8O M AB

9O

M

1 2 3 4 5

7 8 9

+ + + + + +

+ + +

Figure 8. Detection of the single base pair difference in exon 6 of the AB and O alleles by different primers (1:ABO261-AAG, 2: ABO261-

ACG, 3: ABO261-AGG, 4: ABO261-CAG, 5: ABO261-CCG, 6: ABO261-CGG, 7: ABO261-TAG, 8: ABO261-TCG, and 9: ABO261-TGG.). The

arrows indicate target PCR products (135 and 134 bp for the AB and O alleles, respectively). Plus indicates the positive control lane,

where the AB allele was used as a template and the fully matched primers were used. Minus indicates the negative control lane, where

no primers were added to the AB allele. M indicates the 20-bp DNA ladder.




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3.5 Detection of single base pair difference in the

ABO gene using allele-specific primers

Detection of the blood typing is important for paternity

testing, blood infusion, and criminal investigations, andPCR technology enables detection of blood types using DNA

samples that may have been poorly preserved. Table 2

indicates the sequences of the region of exon 6 in the

human ABO gene containing the nucleotides targeted forgenotyping. The 22nd base pair is G/C, both in the A and B

alleles, while the 22nd base pair is A/T in the O allele due to

deletion of the 22nd G/C base pair found in the A and B

alleles. In order to examine genotyping with PCR,

identification of the 22nd base pair in the ABO genes was

carried out using DNA primers (50-TAGGAAGGATG

TCCTCGTGY3Y2G-30

, as given in Table 3, which weredesigned based on the type 1 primer design), forming a G/C

base pair with the A and B alleles but mismatch pairing with

the terminal base of the O allele.

The outline of SNP detection for the human ABO genesis indicated in Fig. 7. Briefly, two-step PCR was used with

the 30 end nucleotide of the forward primers for the 2nd

PCR being located opposite to the SNP nucleotide and with

the adjacent two nucleotides being mismatched with exon 6

sequence (Table 3), resulting in pairings of 50-Y3Y2G-30/50-

CAC-30 for the AB alleles and 50-Y3Y2G-30/50-TAC-30 for the

O allele. The primers have G at the 30 end, which is

complementary to the 22nd C of exon 6 in the AB alleles but

not to the 22nd T in the O allele, and the 2nd (Y2) and the3rd (Y3) nucleotides from the 3

0 end of the primers aremismatched with the 21st A and 20th C, respectively.

Therefore, the PCR products were expected to be less with

the O allele, but significant for the AB allele.

Gel electrophoresis demonstrated that the amplification

products for the AB and O alleles from the 2nd PCR (Fig. 8)

were as expected, with only the AB allele DNA beingamplified efficiently by the primers of ABO261-ACG, AGG,

CCG, TCG, and TGG, and the amplifications by ABO261-

ACG, TCG, and CCG forming the C/A pairing at the 2nd

position from the end were more significant than the other

forward primers (80 nM for ABO261-ACG, 73 nM forABO261-TCG, and 51 nM for ABO261-CCG, respectively, all

of which contain C next to the SNP distinction nucleotide,

G, and in which the last three letters are abbreviated as the

primer name representing the three nucleotides at the 30

end of the primer. For example, the primer ABO261-ACG

has the 30

terminal sequence 50

-ACG-30

). In contrast, theamplification products with O allele DNA using theseprimers were undetectable by the Agilent 2100 Bioanalyzer,

which has a detection sensitivity of about 1.1 nM (Fig. 9).

Consequently, ABO261-ACG, ABO261-TCG, and ABO261-

CCG successfully detected blood type with signal-to-noiseratios of 70.8, 64.6, and 45.1, respectively. Moreover,

ABO261-AAG, CAG, CGG, and TAG also provided less

target amplification product for O allele DNA. These results

are in agreement with those for lambda DNA showing thatprimers forming three consecutive terminal mismatch

pairings prevent amplification. However, when ABO261-AAG, CAG, CGG, and TAG were used, little product was

produced for the AB allele. Importantly, while these fourprimers formed A/A or A/G mismatch at the 2nd position

from the end (50-AAG-30/50-CAC-30, 50-CAG-30/50-CAC-30,50-CGG-30/50-CAC-30, and 50-TAG-30/50-CAC-30, where

underlining indicates the mismatch nucleotides), the

primers of ABO261-ACG, TCG, and CCG, which amplified

the AB allele, formed A/C mismatch at the same position(50-ACG-30/50-CAC-30, 50-TCG-30/50-CAC-30, and 50-CCG-30/50-CAC-30, where underlining indicates the mismatch

nucleotides). A/C mismatch is also formed by the purineand pyrimidine nucleotides, and the purinepyrimidine

pairing may adopt geometry analogous Watson-Crick base

pairing [3033]. It is known that A/C mismatch pairing is

stabilized by the formation of two interstrand hydrogenbonds by protonation of the adenine base. In light of this

structural aspect, it is likely that A/C mismatch pairing in a

DNA duplex results in less distortion of conformation,

presumably leading to less inhibition of the amplificationreaction. Allowance of the A/C mismatch in DNA poly-

merase reactions was also suggested for the terminal A/C

pairing with lambda DNA. On the other hand, the A/A and

G/A mismatches, which are composed of two purine

nucleotides, are supposed to distort the DNA double helixstructure due to their size [3338], and the distortion would

exclude DNA polymerase association. If the model showing

that the geometry at the 2nd position from the 30 end

influences the PCR amplification efficiency is reasonable,the nucleotides specific to SNP are also expected to besuccessful in distinguishing SNP, even when the 2nd

nucleotide of the primer and the corresponding nucleotide

of the template DNA are A and C, respectively. Further-

more, because G/T mismatch is also formed by the purine

and pyrimidine nucleotides and can adopt a geometry

analogous Watson-Crick base pairing like A/C mismatchpair, SNP genotyping may be achieved when the G/T or T/G

mismatch is formed instead of A/C mismatch at the posi-

tion. Primer ABO261-CCG with a C/C mismatch at the

3rd nucleotide position from the end resulted in less

0

20

30

40

50

60

70

80

90

10

AB O

ABO261-ACG

AB O

ABO261-TCG

AB O

ABO261-CCG

C

oncentration

ofamplificationproducts(nM)

Figure 9. Concentrations of the prospective PCR products using

the AB and O alleles as template, and ABO261-ACG, ABO261-

TCG, and ABO261-CCG as an allele-specific primer.




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amplification of the AB allele than did ABO261-ACG or

ABO261-TCG primers in which the A/C or T/C mismatchpairing, respectively, was formed at the same position

(80 nM for ABO261-ACG, 73 nM for ABO261-TCG, and

51 nM for ABO261-CCG, respectively). The 3rd nucleotide,

as well as the 2nd nucleotide from the end may have an

effect on PCR efficiency.The results indicate that the type 1 primers can detect

single base pair differences among the ABO gene alleles.

Although the ability depends on mismatch pairings, espe-

cially at the 2nd position from the 30 end, our results indi-

cate the influences of mismatch pairings and these positionsare useful in designing type 1 primers.

Polymerase is also important for the PCR with allele-

specific DNA primers. The allele-specific type 1 primer that

hybridizes with the template DNA forms mismatched pairsat the 30 end, and the mismatched pairs may prevent DNA

polymerase binding with the primertemplate DNAcomplex. Taq polymerase that has no 30-50 exonuclease

activity was used for these experiments. However, whenusing polymerase that has the 30-50 exonuclease activity

also meant that the pseudopositive problem may occurbecause such a polymerase may degrade the mismatched

nucleotides. The use of the DNA polymerase without the

30-50 exonuclease activity is an important consideration for

SNP detection.

4 Concluding remarks

To investigate the allele-specific primer DNA sequences that

can eliminate the pseudopositive problem, PCR experi-

ments were carried out systematically using lambda DNA asa template and the DNA primers with different numbers of

and different types of mismatch pairings near the 3 0 end.

The present findings showed that primers forming a single

mismatch pairing at the 30 end of these primers gave theamplification products (primer nos. 24 in Fig. 2), indicat-

ing that only a single terminal mismatch is insufficient for

inhibiting the amplification, as has also been reported

previously [2325]. On the other hand, amounts of the PCR

product obtained for DNA primers forming two consecutive

mismatch pairings at the end strongly depended on themismatch type and number of amplification cycles (primer

nos. 513 in Fig. 2). In particular, the primers forming a

mismatched pairing next to the A/C or T/C pair at the endprovided moderate amounts of the amplification productafter 30 amplification cycles, indicating the importance of

control of the PCR cycle number for the regulation in the

amount of product. In contrast with the two-mismatch

primers forming the A/C or T/C mismatch pairing at the

end, less PCR product was observed for the primers formingtwo consecutive C/C mismatch pairings at the end, even

after the 30 cycles. Because the primer forming the G/C

pairing next to the C/C mismatch pairing at the end gave

amplification product, the genotyping of G and C in analyteDNA can be realized by using these primers.

As for other SNP genotypings, such as those formingA/C and T/C mismatch pairings, two consecutive mismat-

ches at the end seem inefficient to discriminate

the sequences. However, primers forming three consecutive

mismatches at the end show less target PCR productregardless of the mismatch type, even after the 30 amplifi-

cation cycles (Fig. 3). When the 30

end nucleotide of the type1 primer was mismatched with the template DNA, threeconsecutive mismatches at the 30 end were formed, while

moderate amounts of PCR product were observed for the

formation of the terminal G/C pairing (Fig. 6). Thus, the

type 1 primer is promising for PCR cycle-independent SNPdetection within at the most 30 amplification cycles, while

the detection possibly fails after larger amplification cycles

because pseudopositive signals from the primers with three

consecutive mismatches become large and non-negligible.Examinations of human ABO genes also demonstrated that

type 1 primer design was useful for detecting single basepair difference in the gene sequences with high signal-to-

noise ratios.In this paper, systematic PCR experiments were carried

out to investigate allele-specific primers without the pseudo-positive problem. Although direct sequence analyses such as

Sanger method [39], pyrosequencing method [2022], and

single-base extension method [40] can also be useful for the

SNP detection, the method using PCR with allele-specificprimers have great advantages in time, cost, and a handling

with ease even though the design of allele-specific primers for

every new SNP analysis is needed. Examinations of human

ABO genes gave us important information to design allele-

specific primers. The results showed that the primers form-

ing the A/C mismatch pairing next to the G/C pairing at the

30

end supported greater PCR amplification than did the A/Aor the G/A mismatch pair, which indicates the importance of

the geometry of the mismatch pair at the 2nd position from

the 30 end. When the DNA sequence design of templates and

primers are optimized, type 1 primer design may also besuitable for identifying genes other than the human ABO

gene. Consequently, type 1 primers can be used to detect SNP

with less occurrence of the pseudopositive problem.

The authors declare no conflict of interest.

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