Modified nucleic acid amplification primers

The present invention provides modified primers for use in the amplification of a nucleic acid sequence. Amplifications carried out using the modified primers result in less non-specific amplification product, in particular, primer dimer, and a concomitant greater yield of the intended amplification product compared to amplifications carried out using unmodified primers.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the field of molecular biology and nucleic 
acid chemistry. More specifically, it relates to methods and reagents for 
improving the yield of nucleic acid amplification reactions. The 
invention, therefore, has applications in any field in which nucleic acid 
amplification is used. 
2. Description of Related Art 
The invention of the polymerase chain reaction (PCR) made possible the in 
vitro amplification of nucleic acid sequences. PCR is described in U.S. 
Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; Saiki et al., 1985, Science 
230:1350-1354; Mullis et al., 1986, Cold Springs Harbor Symp. Quant. Biol. 
51:263-273; and Mullis and Faloona, 1987, Methods Enzymol. 155:335-350; 
each of which is incorporated herein by reference. The development and 
application of PCR are described extensively in the literature. For 
example, a range of PCR-related topics are discussed in PCR 
Technology--principles and applications for DNA amplification, 1989, (ed. 
H. A.Erlich) Stockton Press, New York; PCR Protocols: A guide to methods 
and applications, 1990, (ed. M. A. Innis et al.) Academic Press, San 
Diego; and PCR Strategies, 1995, (ed. M. A. Innis et al.) Academic Press, 
San Diego; each of which is incorporated herein by reference. Commercial 
vendors, such as Perkin Elmer (Norwalk, Conn.), market PCR reagents and 
publish PCR protocols. 
Since the original publication of nucleic acid amplification, various 
primer-based nucleic acid amplification methods have been described 
including, but are not limited to, Ligase Chain Reaction (LCR, Wu and 
Wallace, 1989, Genomics 4:560-569 and Barany, 1991, Proc. Natl. Acad. Sci. 
USA 88:189-193); Polymerase Ligase Chain Reaction (Barany, 1991, PCR 
Methods and Applic. 1:5-16); Gap-LCR (PCT Patent Publication No. WO 
90/01069); Repair Chain Reaction (European Patent Publication No. 439,182 
A2), 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177; 
Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878; PCT Patent 
Publication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238). All of 
the above references are incorporated herein by reference. A survey of 
amplification systems is provided in Abramson and Myers, 1993, Current 
Opinion in Biotechnology 4:41-47, incorporated herein by reference. 
Specificity of primer-based amplification reactions depends on the 
specificity of primer hybridization. Under the elevated temperatures used 
in a typical amplification, the primers hybridize only to the intended 
target sequence. However, amplification reaction mixtures are typically 
assembled at room temperature, well below the temperature needed to insure 
primer hybridization specificity. Under such less stringent conditions, 
the primers may bind non-specifically to other only partially 
complementary nucleic acid sequences or to other primers and initiate the 
synthesis of undesired extension products, which can be amplified along 
with the target sequence. Amplification of non-specific primer extension 
products can compete with amplification of the desired target sequences 
and can significantly decrease the efficiency of the amplification of the 
desired sequence. 
One frequently observed type of non-specific amplification product is a 
template independent artifact of amplification reactions referred to as 
"primer dimer". Primer dimer is a double-stranded fragment whose length 
typically is close to the sum of the two primer lengths and appears of 
occur when one primer is extended over the other primer. The resulting 
concatenation forms an undesired template which, because of its short 
length, is amplified efficiently. 
Non-specific amplification can be reduced by reducing the formation of 
primer extension products prior to the start of the reaction. In one 
method, referred to as a "hot-start" protocol, one or more critical 
reagents are withheld from the reaction mixture until the temperature is 
raised sufficiently to provide the necessary hybridization specificity. In 
this manner, the reaction mixture cannot support primer extension during 
the time that the reaction conditions do not insure specific primer 
hybridization. 
Manual hot-start methods, in which the reaction tubes are opened after the 
initial high temperature incubation step and the missing reagents are 
added, are labor intensive and increase the risk of contamination of the 
reaction mixture. Alternatively, a heat sensitive material, such as wax, 
can be used to separate or sequester reaction components, as described in 
U.S. Pat. No. 5,411,876, incorporated herein by reference, and Chou et 
al., 1992, Nucl. Acids Res. 20(7):1717-1723, incorporated herein by 
reference. In these methods, a high temperature pre-reaction incubation 
melts the heat sensitive material, thereby allowing the reagents to mix. 
Another method of reducing the formation of primer extension products prior 
to the start of the reaction relies on the heat-reversible inhibition of 
the DNA polymerase by DNA polymerase-specific antibodies, as described in 
U.S. Pat. No. 5,338,671, incorporated herein by reference. The antibodies 
are incubated with the DNA polymerase in a buffer at room temperature 
prior to the assembly of the reaction mixture in order to allow formation 
of the antibody-DNA polymerase complex. Antibody inhibition of DNA 
polymerase activity is inactivated by a high temperature pre-reaction 
incubation. A disadvantage of this method is that the production of 
antibodies specific to the DNA polymerase is expensive and time-consuming, 
especially in large quantities. Furthermore, the addition of antibodies to 
a reaction mixture may require redesign of the amplification reaction. 
The formation of extension products prior to the start of the reaction can 
also be inhibited by the addition to the reaction of a single-stranded 
binding protein, which non-covalently binds to the primers in a 
heat-reversible manner and inhibits primer extension by preventing 
hyridization. 
Non-specific amplification also can be reduced by enzymatically degrading 
extension products formed prior to the start of the reaction using the 
methods described in U.S. Pat. No. 5,418,149, which is incorporated herein 
by reference. The degradation of newly-synthesized extension products is 
achieved by incorporating into the reaction mixture dUTP and UNG, and 
incubating the reaction mixture at 45-60.degree. C. prior to carrying out 
the amplification reaction. Primer extension results in the formation of 
uracil-containing DNA, which is degraded by UNG under the 
pre-amplification conditions. A disadvantage of this method is that the 
degradation of extension product competes with the formation of extension 
product and the elimination of non-specific primer extension product is 
likely to be less complete. An advantage of this method is that 
uracil-containing DNA introduced into the reaction mixture as a 
contamination from a previous reaction is also degraded and, thus, the 
method also reduces the problem of contamination of a PCR by the amplified 
nucleic acid from previous reactions. 
Conventional techniques of molecular biology and nucleic acid chemistry, 
which are within the skill of the art, are fully explained fully in the 
literature. See, for example, Sambrook et al., 1989, Molecular Cloning--A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New 
York; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid 
Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, 
Methods in Enzymology (Academic Press, Inc.), all of which are 
incorporated herein by reference. All patents, patent applications, and 
publications mentioned herein, both supra and infra, are incorporated 
herein by reference. 
SUMMARY OF THE INVENTION 
The present invention provides covalently modified oligonucleotide primers 
for the in vitro amplification of nucleic acid sequences. Use of the 
modified primers of the invention results in a reduction in non-specific 
amplification, especially primer dinner formation, and/or a concomitant 
increase in the yield of the intended target relative to an amplification 
carried out with unmodified primers. 
A variety of modifier moeities are envisioned which possess the following 
properties: 
1. interfere with, but not prevent, Watson-Crick base pairing of the 
modified base with the complementary base; 
2. interfere with, but not prevent, extension of the modified primer; and 
3. allow synthesis of a strand complementary to the extension product of 
the modified primer. 
One aspect of the invention relates to an oligonucleotide primer for the 
amplification of a nucleic acid sequence, having the general structure: 
##STR1## 
wherein S.sub.1 represents a first sequence of nucleotides between about 5 
and about 50 nucleotides in length; 
wherein S.sub.2 represents a second sequence between one and three 
nucleotides in length; 
wherein N represents a nucleotide that which contains a purine or 
pyrimidine base that contains an exocyclic amine; 
wherein R represents a modifier group, wherein R is covalently bound to N 
through the exocyclic amine, and and wherein R has the structure: 
##STR2## 
wherein R.sub.1 and R.sub.2 represent independently hydrogen, a C.sub.1 
-C.sub.10 alkyl group, an alkoxy group, a phenyl group, a phenoxy group, a 
substituted phenyl group, a napthyl group, or a substituted napthyl group. 
Alkyl groups may be branched or unbranched. 
In a preferred embodiment, N is a modified conventional nucleotide, in 
which case N is a modified adenosine, cytidine, or guanosine, and the 
modifier moiety is covalently attached to the exocyclic amine of an 
adenine, guanine, or cytosine base. In a more preferred embodiment, N is a 
modified adenosine. 
In a preferred embodiment, R is a 2-napthylmethyl group; a benzyl group; or 
a substituted benzyl group. Preferred substituted benzyl groups have the 
structure: 
##STR3## 
wherein R.sub.3 represent a C.sub.1 -C.sub.6 branched or unbranched alkyl 
group, more preferably a C.sub.1 -C.sub.4 branched or unbranched alkyl 
group, a methoxy group, or a nitro group. Preferably, R.sub.3 is attached 
in the para position. 
In more preferred embodiment, R is a benzyl, p-methylbenzyl, 
p-tert-butylbenzyl, p-methoxybenzyl, or 2-napthylmethyl group. 
Another aspect of the invention relates to amplification primers which are 
modified by the photo-labile covalent attachment of a modifier group, 
which results in a partial or complete inhibition of primer extension. The 
photo-labile modifier may be bound either to the exocyclic amine, as in 
the modified nucleotides described above, or to the ring nitrogen. In one 
embodiment, at least one nitrobenzyl group is attached to the exocyclic 
amine of an adenine, guanine, or cytosine base of the 3' terminal 
nucleotide. 
Another aspect of the invention is a pair or set of primers, wherein at 
least one of the primers is modified as described above. In a prefered 
embodiment, both members of a pair, or all members of a set, of primers 
are modified. 
Another aspect of the invention relates to methods for amplifying nucleic 
acid which comprise carrying out an amplificaton reaction using the 
modified primers of the invention. 
Another aspect of the invention relates to methods for amplifying a target 
nucleic acid which comprise carrying out an amplificaton reaction using 
the photo-labile modified primers of the invention, wherein the reaction 
mixture is irradiated with light sufficient to remove the modifier group 
and allow formation of primer extension products. In one embodiment of the 
invention, the irradiation is carried out as a separate step, prior to the 
start of the amplification reaction, but after the reaction mixture has 
been heated to a temperature greater than about 50.degree. C. In other 
embodiments, the irradiation step is combined with a preliminary step of 
the amplification process, such as the reverse transcription step of in an 
RNA amplification reaction, or the initial denaturation step in a DNA 
amplification reaction. 
Another aspect of the invention relates to kits for the in vitro 
amplification of nucleic acid sequences, which kits comprise a pair of 
primers in which at least one of the primers is modified as described 
herein. The kits of the present invention also can include one or more 
amplification reagents, e.g., a nucleic acid polymerase or ligase, 
nucleoside triphosphatase, and suitable buffers.

DETAILED DESCRIPTION OF THE INVENTION 
To aid in understanding the invention, several terms are defined below. 
The terms "nucleic acid" and "oligonucleotide" refer to 
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to 
polyribonucleotides (containing D-ribose), and to any other type of 
polynucleotide which is an N glycoside of a purine or pyrimidine base, or 
modified purine or pyrimidine base. There is no intended distinction in 
length between the terms "nucleic acid" and "oligonucleotide", and these 
terms will be used interchangeably. These terms refer only to the primary 
structure of the molecule. Thus, these terms include double- and 
single-stranded DNA, as well as double- and single-stranded RNA. 
The term "conventional", in reference to nucleic acid bases, nucleosides, 
or nucleotides, refers to those which occur naturally in the 
polynucleotide being described. The four conventional (also referred to as 
major) deoxyribonucleotides of DNA contain the purine bases adenine and 
guanine and the pyrimidine bases cytosine and thymine. The four 
conventional ribonucleotides of RNA contain the purine bases adendine and 
guanine and the pyrimidine bases cytosine and uracil. In addition to the 
above conventional or common bases, a number of other puring and 
pyrimidine derivatives, called rare or minor bases, occur in small amounts 
in some nucleic acids. As used herein, "unconventional", in reference to 
nucleic acid bases, nucleosides, or nucleotides, refers to rare or minor 
nucleic acid bases, nucleosides, or nucleotides, and modifications, 
derivations, or analogs of conventional bases, nucleosides, or 
nucleotides, and includes synthetic nucleotides having modified base 
moieties and/or modified sugar moieties (see, Protocols for 
Oligonucleotide Conjugates, Methods in Molecular Biology, Vol 26, (Sudhir 
Agrawal, Ed., Humana Press, Totowa, N.J., (1994)); and Oligonucleotides 
and Analogues, A Practical Approach (Fritz Eckstein, Ed., IRL Press, 
Oxford University Press, Oxford); both incorporated herein by reference). 
Oligonucleotides can be prepared by any suitable method, including, for 
example, cloning and restriction of appropriate sequences and direct 
chemical synthesis by a method such as the phosphotriester method of 
Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of 
Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite 
method of Beaucage et al., 1981, Tetrahedron Lett. 22:1859-1862; and the 
solid support method of U.S. Pat. No. 4,458,066, each incorporated herein 
by reference. A review of synthesis methods is provided in Goodchild, 
1990, Bioconiugate Chemistry 1(3):165-187, incorporated herein by 
reference. 
The term "base pairing", also referred to in the art as "Watson-Crick base 
pairing", refers to the well known hydrogen bonding of complementary base 
pairs adenine-thymine and guanine-cytosine in a double stranded DNA 
structure, adenine-uracil and guanine-cytosine in a RNA/DNA hybrid 
molecule, and to analogous bonding of unconventional nucleotide pairs. 
The term "hybridization" refers the formation of a duplex structure by two 
single-stranded nucleic acids due to complementary base pairing. 
Hybridization can occur between fully complementary nucleic acid strands 
or between "substantially complementary" nucleic acid strands that contain 
minor regions of mismatch. Conditions under which only fully complementary 
nucleic acid strands will hybridize are referred to as "stringent 
hybridization conditions" or "sequence-specific hybridization conditions". 
Stable duplexes of substantially complementary sequences can be achieved 
under less stringent hybridization conditions; the degree of mismatch 
tolerated can be controlled by suitable adjustment of the hybridization 
conditions. Those skilled in the art of nucleic acid technology can 
determine duplex stability empirically considering a number of variables 
including, for example, the length and base pair concentration of the 
oligonucleotides, ionic strength, and incidence of mismatched base pairs, 
following the guidance provided by the art (see, e.g., Sambrook et al., 
1989, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y.; and Wetmur, 1991, Critical Review in 
Biochem. and Mol. Biol. 26(3/4):227-259; both incorporated herein by 
reference). 
The term "primer" refers to an oligonucleotide capable of acting as a point 
of initiation of DNA synthesis under conditions in which synthesis of a 
primer extension product complementary to a nucleic acid strand is 
induced, i.e., either in the presence of four different nucleoside 
triphosphates and an agent for extension (e.g., a DNA polymerase or 
reverse transcriptase) in an appropriate buffer and at a suitable 
temperature. As used herein, the term "primer" is intended to encompass 
the oligonucleotides used in ligation-mediated amplification processes, in 
which one oligonucleotide is "extended" by ligation to a second 
oligonucleotide which hybridizes at an adjacent position. Thus, the term 
"primer extension", as used herein, refers to both the polymerization of 
individual nucleoside triphosphates using the primer as a point of 
initiation of DNA synthesis and to the ligation of two primers to form an 
extended product. 
A primer is preferably a single-stranded DNA. The appropriate length of a 
primer depends on the intended use of the primer but typically ranges from 
6 to 50 nucleotides. Short primer molecules generally require cooler 
temperatures to form sufficiently stable hybrid complexes with the 
template. A primer need not reflect the exact sequence of the template 
nucleic acid, but must be sufficiently complementary to hybridize with the 
template. The design of suitable primers for the amplification of a given 
target sequence is well known in the art and described in the literature 
cited herein. 
Primers can incorporate additional features which allow for the detection 
or immobilization of the primer but do not alter the basic property of the 
primer, that of acting as a point of initiation of DNA synthesis. For 
example, primers may contain an additional nucleic acid sequence at the 5' 
end which does not hybridize to the target nucleic acid, but which 
facilitates cloning of the amplified product. The region of the primer 
which is sufficiently complementary to the template to hybridize is 
refered to herein as the hybridizing region. 
The terms "target, "target sequence", "target region", and "target nucleic 
acid" refer to a region or subsequence of a nucleic acid which is to be 
amplified. 
As used herein, a primer is "specific" for a target sequence if the number 
of mismatches present between the primer sequence and the target sequence 
is less than the number of mismatches present between the primer sequence 
and non-target sequences which may be present in the sample. Hybridization 
conditions can be chosen under which stable duplexes are formed only if 
the number of mismatches present is no more than the number of mismatches 
present between the primer sequence and the target sequence. Under such 
conditions, the primer can form a stable duplex only with a target 
sequence. Thus, the use of target-specific primers under suitably 
stringent amplification conditions enables the specific amplification of 
those target sequences which contain the target primer binding sites. The 
use of sequence-specific amplification conditions enables the specific 
amplification of those target sequences which contain the exactly 
complementary primer binding sites. 
The term "non-specific amplification" refers to the amplification of 
nucleic acid sequences other than the target sequence which results from 
primers hybridizing to sequences other than the target sequence and then 
serving as a substrate for primer extension. The hybridization of a primer 
to a non-target sequence is referred to as "non-specific hybridization" 
and can occur during the lower temperature, reduced stringency, 
pre-amplification conditions. 
The term "primer dimer" refers to template-independent non-specific 
amplification product which results from primer extensions wherein another 
primer serves as a template. Although primer dimer frequently appears to 
be a concatamer of two primers, i.e., a dimer, concatamers of more than 
two primers also occur. The term "primer dimer" is used generically herein 
to encompasses template-independent non-specific amplification product. 
The term "reaction mixture" refers to a solution containing reagents 
necessary to carry out a given reaction. An "amplification reaction 
mixture", which refers to a solution containing reagents necessary to 
carry out an amplification reaction, typically contains oligonucleotide 
primers and a DNA polymerase or ligase in a suitable buffer. A "PCR 
reaction mixture" typically contains oligonucleotide primers, a 
thermostable DNA polymerase, dNTP's, and a divalent metal cation in a 
suitable buffer. A reaction mixture is referred to as complete if it 
contains all reagents necessary to enable the reaction, and incomplete if 
it contains only a subset of the necessary reagents. It will be understood 
by one of skill in the art that reaction components are routinely stored 
as separate solutions, each containing a subset of the total components, 
for reasons of convenience, storage stability, or to allow for 
application-dependent adjustment of the component concentrations, and, 
that reaction components are combined prior to the reaction to create a 
complete reaction mixture. Furthermore, it will be understood by one of 
skill in the art that reaction components are packaged separately for 
commercialization and that useful commercial kits may contain any subset 
of the reaction components which includes the modified primers of the 
invention. 
All patents, patent applications, and publications mentioned herein, both 
supra and infra, are incorporated herein by reference. 
Modified Primers 
The amplification primers of the invention are modified by the covalent 
attachment of a group to one of the four nucleotides at the 3'-terminal 
end of the primer. In one embodiment, a modified primer of the invention 
consists of a nucleic acid sequence having the general structure: 
##STR4## 
wherein S.sub.1 represents a first sequence of nucleotides between about 5 
and about 50 nucleotides in length; 
wherein S.sub.2 represents a second sequence between one and three 
nucleotides in length; 
wherein N represents a nucleotide that which contains a purine or 
pyrimidine base that contains an exocyclic amine; 
wherein R represents a modifier group, wherein R is covalently bound to N 
through the exocyclic amine, and wherein R has the structure described 
below. 
As shown in the examples, the effect of the modification is maximized when 
the modification is to the 3' terminal nucleotide. Thus, preferably, the 
primer contains a modified 3' terminal nucleotide. 
The modified nucleotide is selected from those whose base contains an 
exocyclic amine that is involved in the base pairing of the nucleotide 
with its complementary nucleotide. Typically, primers are DNA containing 
only conventional nucleotides. Of the four conventional nucleotide bases 
found in DNA, adenine, guanine, and cytosine contain an exocyclic primary 
amine which is involved in base pairing with the complementary base. In 
the preferred aspect of the invention, the primer is modified by the 
attachment of a single modifier group to the exocyclic amine, substituting 
for one of the two hydrogen of the amine group which, in the unmodified 
base, are capable of being involved in base pairing. The structures of the 
modified nucleotides containing a modified adenine, guanine, and cytosine 
base, respectively, are shown below. 
##STR5## 
where S represents the sugar, and R represents the modifier group. 
The present invention is not limited to primers consisting of conventional 
nucleotides. Any nucleotide analog in which the base moiety contains an 
exocyclic primary amine which is involved in base pairing with a 
complementary base is modifiable as described herein. Examples of 
unconventional nucleotides include 3-methyladenine, 7-methylguanine, 
3-methylguanine, 5-methyl cytosine, and 5-hydroxymethyl cytosine. 
The modifier group limits the ability of the modified base to participate 
in hydrogen bonding because the modifier substitutes for one hydrogen 
atom. The remining hydrogen atom still can participate in hydrogen 
bonding. The modifiers can therefore influence both the kinetics and 
thermodynamics of hybridization. A variety of modifier groups are 
envisioned which possess the following properties: 
1. interfere with, but not prevent, Watson-Crick base pairing of the 
modified base with the complementary base; 
2. interfere with, but not prevent, extension of the modified primer; and 
3. allow synthesis of a strand complementary to the extension product of 
the modified primer. 
The modifier group sterically interferes with base pairing and, thus, with 
primer extension. Thus, the physical bulk of the modifier influences the 
degree of interference with hybridization. When a modified adenosine or 
cytidine nucleotide is incorporated into a double-stranded nucleic acid, 
the modifier group protrudes into the central space of the major groove. 
Consequently, even relatively large modifier groups should cause little 
steric perturbation of the duplex structure. However, suitable modifiers 
are not so large such that hydrogen bonding is prevented or enzymatic 
extension of the 3'-hydroxyl of the primer is prevented. When the modified 
guanosine nucleotide is incorporated into a double-stranded nucleic acid, 
the modifier group protrudes into the minor groove, which provides less 
room to accomodate the bulk of the modifier group. Consequently, smaller 
modifier groups are prefered for attachment to a guanine base. 
Primer extension products, which are used as templates in subsequent 
amplification cycles, contain the modified base introduced by the primer. 
The modifier group is chosen such that the presence of the modified base 
in the template does not cause termination of primer extension or 
inhibition of primer extension. Preferrably, the nature of the modifier 
group should not give rise to mutagenic events whereby the identity of the 
modified base is lost on replication of a primer-derived template. The 
effect of the modified base in the template on primer extension can be 
routinely tested following the guidance provided herein and in the art 
(see, for example, Gniazdowski and Cera, 1996, Chem. Rev. 96:619-634, 
incorporated herein by reference). 
Modifier groups, R, which satisfy the above properties are suitable for use 
in the methods of the present invention. Prefered modifier groups have the 
structure: 
##STR6## 
wherein R.sub.1 and R.sub.2 represent independently hydrogen, a C.sub.1 
-C.sub.10 alkyl group, an alkoxy group, a phenyl group, a phenoxy group, a 
substituted phenyl group, a napthyl group, or a substituted napthyl group. 
Alkyl groups may be branched or unbranched. Larger alkyl groups, up to at 
least C.sub.20, may also be used. 
In a preferred embodiment, R is a 2-napthylmethyl group; a benzyl group; or 
a substituted benzyl group. Preferred substituted benzyl groups have the 
structure: 
##STR7## 
wherein R.sub.3 represent a C.sub.1 -C.sub.6 branched or unbranched alkyl 
group, more preferably a C.sub.1 -C.sub.4 branched or unbranched alkyl 
group, a methoxy group, or a nitro group. Preferably, R.sub.3 is attached 
in the para position. 
Particularly preferred modifier groups are shown below: 
##STR8## 
A number of particular modifier groups are described in the examples. In 
general, empirical selection of a particular suitiable modifier group from 
the class of compounds described can be carried out routinely by one of 
skill in the art following the guidance provided herein. Preferably, 
suitability of a particular group is determined empirically by using the 
modified primers in an amplification reaction. Successful amplification 
indicates both that the modified base does not totally inhibit primer 
extension, and that presence of the modified base in a primer derived 
template does not cause termination of primer extension. The reduction of 
primer dimer is determined as described in the examples. 
Theory of operation 
In each cycle of a primer-based amplification, primers are annealed to 
target nucleic acid, and the primers are enzymatically extended. The 
process is repeated typically between 25 and 40 times. The specificity of 
the amplification depends on the specificity of the primer hybridization 
step. Primer sequences and reaction conditions are selected such that the 
primers form stable hybridization duplexes only with the complementary 
sequences present in the intended target nucleic acid sequence. 
It is believed that non-specific amplification occurs when an unstable, 
transient hybridization duplex is formed between a primer and a non-target 
molecule, possibly another primer, in which the 3' end of the primer is 
momentarily paired with a complementary base in the other molecule. 
Initial primer extension results in the formation of complementary 
sequence which stabilizes the duplex and allows further extension. 
While not being constrained by the theory, it is believed that the stable 
modified primers of the present invention, which remain modified 
throughout the reaction, reduce non-specific amplification by increasing 
the time required for the initial primer extension to occur. The modifier 
group, when rotated towards the complementary base, sterically hinders 
base pairing. However, rotation of the amine into a configuration in which 
the hydrogen is directed towards the complementary base permits normal 
base pairing. Primer extension, which depends on the formation of matching 
base pairs at the 3' end of the primer, is delayed until the amine group 
has rotated into a permissive position and base pairing has occurred. The 
additional time required for the rotation into a permissive configuration 
reduces the likelihood that an unstable, transient hybridization duplex, 
such as between primers under pre-reaction conditions, will exist for a 
sufficient time to permit primer extension. 
In contrast, primer-target hybridization duplexes are sufficiently stable 
under the primer hybridization condition used in an amplification so as to 
provide time for the rotation of the amine into a configuration which 
allows base pairing with the complementary base. Following hybridization, 
primer extension appears not to be affected. Thus, the modification does 
not significantly inhibit primer extension under the amplification 
conditions, but does decrease the probability of extension of primers 
involved in unstable, transient duplexes formed with non-target sequences 
under the pre-amplification conditions. 
Primers with a Photo-labile Modification 
In an alternative embodiment of the invention, primers are modified with 
one or more photo-labile groups which can be removed by exposure to light 
after the reaction has reach the high-temperature reaction conditions 
which insure specificity. Becauses the modifier is removed prior to primer 
extension, the modified primer need not be extendable prior to removal of 
the group. Examples of photolabile modifiers which can be used in the 
methods of the present invention are described in Pillai, 1980, 
"Photoremovable Protecting Groups in Organic Synthesis", Synthesis: 1-26, 
incorporated herein by reference. 
Preferably, the photo-labile primers of the invention are modified at the 
3' terminal nucleotide by the attachment of one or two o-nitrobenzyl 
groups: 
##STR9## 
In primers modified by the attachment of a single nitrobenzyl group to the 
exocyclic primary amine of a base moiety, the resulting secondary amine 
still can participate in base pairing if the amine group is rotated such 
that the remaining hydrogen is oriented towards the complementary base. As 
described in the examples, these primers can be used in an amplification 
either with or without removal by irradiation with UV light. 
Primers modified by the attachment of a two nitrobenzyl groups to the 
exocyclic amine of the base cannot be extended. The inhibition presumably 
results from the inability of the modified base to undergo base pairing, 
which is precluded because both hydrogens of the exocyclic amine are 
replaced by bulky nitrobenzyl groups. The use of primers modified with two 
nitrobenzyl groups in an amplification, in which the reaction mixture was 
exposed to UV light for a time sufficient to remove the nitrobenzyl 
groups, thereby allowing primer extension to take place, is described in 
the examples. 
In an alternative embodiment, the modifier group is attached to the ring 
nitrogen. Primers modified by the attachment of a nitrobenzyl group to the 
ring nitrogen of the base cannot be extended due to the inability of the 
modified base to undergo base pairing. Removal of the nitrobenzyl groups 
by exposure to UV light allows primer extension to take place. 
Use of the photo-labile primers which cannot be extended until the modifier 
group is removed essentially provides a "hot-start" amplification. Primer 
extension is inhibited during the non-specific pre-reaction conditions. 
The reaction is irradiated and the primers deblocked only after the 
reaction temperature has been raised to a temperature which insures 
reaction specificity. 
Synthesis of Modified Primers 
Synthesis of the modified primers is carried out using standard chemical 
means well known in the art. Methods for the introduction of these 
modifiers can be divided into four classes. 
1. The modifier can be introduced by use of a modified nucleoside as a DNA 
synthesis support. 
2. The modifier can be introduced by use of a modified nucleoside as a 
phosphoramidite. 
3. The modifier can be introduced by the use of a reagent during DNA 
synthesis. (e.g., benzylamine treatment of a convertible amidite when 
incorporated into a DNA sequence). 
4. Post-synthetic modification. The modifier can be introduced as a 
reactive reagent when contacted with synthetic DNA. 
The synthesis of particular modified primers is described in the examples. 
Additional modified primers can be synthesized using standard synthesis 
methods in an analogous manner. 
Preferably, modified primers are synthesized using a derivatized controlled 
pore glass (CPG) synthesis support. A general reaction scheme for the 
synthesis of derivatized dA CPG is shown in FIG. 6. Particular modifier 
groups can be added by use of the appropriate alkyl-halide, benzyl-halide, 
substituted benzyl halide, methylnapthyl-halide, or substituted 
methylnapthyl-halide alkylating agent. The syntheses of the benzyl- and 
p-ert-butylbenzyl-dA CPG describes in Examples 1 and 2 follow the scheme 
shown in FIG. 6. 
Alkylation of the exocyclic amino group can be carried out using methods 
analogous to the methylation described in Griffin and Reese, 1963, 
Biochim. Acta 68:185-192, incorporated herein by reference. Additional 
synthesis methods are described in Aritoma el al. 1995, J. Chem. Soc. 
Perkin Trans. 1: 1837-1844, which is incorporated herein by reference. 
Amplifications using Modified Primers 
The methods of the present invention comprise carrying out a primer-based 
amplification using the modified primers of the present invention. In 
general, the modified primers can be substituted for unmodified primers 
containing the same nucleotide sequence in a primer-based amplification 
with no change in the amplification reaction conditions. Of course, one of 
skill in the art will recognize that routine minor re-optimization of the 
reaction conditions may be benificial in some reactions. 
In a preferred embodiment, the modified primers of the present invention 
are used in the polymerase chain reaction (PCR). However, the invention is 
not restricted to any particular amplification system. The modified 
primers of the present invention can be used in any primer-based 
amplification system in which primer dimer or non-specific amplification 
product can be formed. Examples include the amplification methods 
described in the references cited above. As other systems are developed, 
those systems may benefit by practice of this invention. 
The methods of the present invention are suitable for the amplification of 
either DNA or RNA. For example, the amplification of RNA using a reverse 
transcription/polymerase chain reaction (RT-PCR) is well known in the art 
and described in U.S. Pat. Nos. 5,322,770 and 5,310,652, Myers and 
Gelfand, 1991, Biochemistry 30(31):7661-7666, Young et al., 1993, J. Clin. 
Microbiol. 31(4):882-886, and Mulder et al., 1994, J. Clin. Microbiol. 
32(2):292-300, each incorporated herein by reference. 
In a primer-based amplification, primer extension is carried out typically 
at an elevated temperature using a thermostable enzyme such as a 
thermostable DNA polymerase. The enzyme initiates synthesis at the 3' end 
of the primer and proceeds in the direction towards the 5' end of the 
template until synthesis terminates. Purified thermostable DNA polymerases 
useful in amplification reactions are well known in the art and include, 
but are not limited to, the enzymes described in U.S. Pat. No. 4,889,818; 
U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,352,600; U.S. Pat. No. 5,491,086; 
WO 91/09950; WO 92/03556, WO 92/06200; WO 92/06202; WO 92/09689; and U.S. 
Pat. No. 5,210,036; each incorporated herein by reference. A review of 
thermostable DNA polymerases is provided in Abramson, 1995, in PCR 
Strategies, (ed. M. A. Innis et al.), pp 39-57, Academic Press, San Diego, 
incorporated herein by reference. 
In a preferred embodiment, particularly for the amplification of DNA, the 
amplification is carried out using a reversibly inactivated enzyme as 
described in copending U.S. patent application Ser. Nos. 08/680,283 and 
08/684,108, which both represent regular U.S. filings of provisional 
application No. 60/002,673, each incorporated herein by reference. The use 
of a reversibly inactivated enzyme, which is re-activated under the high 
temperature reaction conditions, further reduces non-specific 
amplification by inhibiting primer extension prior to the start of the 
reaction. A reversibly inactivated thermostable DNA polymerase, developed 
and manufactured by Hoffmann-La Roche (Nutley, N.J.) and marketed by 
Perkin Elmer (Norwalk, Conn.), is described in Birch et al., 1996, Nature 
381(6581):445-446, incorporated herein by reference. 
The effect of the modifier group on the ability of the enzyme to extend the 
primer depends, in part, on the particular enzyme used and, in part, on 
the reaction conditions selected. For example, Tth DNA polymerase is more 
permissive when Mn.sup.2+ is used as the divalent cation, as in some RNA 
amplifications, rather that Mg.sup.2+. One of skill will recognize that in 
the routine selection of a suitable modifier group, the enzyme and 
reaction conditions will be considered. 
Sample preparation methods suitable for amplification reactions are well 
known in the art and fully described in the literature cited herein. The 
particular method used is not a critical part of the present invention. 
One of skill in the art can optimize reaction conditions for use with the 
known sample preparation methods. 
Methods of analyzing amplified nucleic acid are well known in the art and 
fully described in the literature cited herein. The particular method used 
is not a critical part of the present invention. One of skill in the art 
can select a suitable analysis method depending on the application. 
A preferred method for analyzing an amplification reaction is by monitoring 
the increase in the total amount of double-stranded DNA in the reaction 
mixture, as described in in Higuchi et al., 1992, Bio/Technology 
10:413-417; Higuchi et al., 1993, Bio/Technology 11:1026-1030; European 
Patent Publication No. 512,334; and copending U.S. patent application Ser. 
No. 08/266,061; each incorporated herein by reference. In this method, 
referred to herein as "kinetic PCR", the detection of double-stranded DNA 
relies on the increased fluorescence that ethidium bromide (EtBr) and 
other DNA binding labels exhibit when bound to double-stranded DNA. The 
amplification is carried out in the presence of the label. The increase of 
double-stranded DNA resulting from the synthesis of target sequences 
results in a detectable increase in fluorescence, which is monitored 
during the amplification. Thus, the methods enable monitoring the progress 
of an amplification reaction. 
In a kinetic PCR, the measured fluorescence depends on the total amount of 
double-stranded DNA present, whether resulting from non-specific 
amplification or from amplification of the target sequence. Monitoring the 
fluorescence allows measurement of the increase in the total amount of 
double-stranded DNA, but the increase resulting from amplification of the 
target sequence is not measured independently from the increase resulting 
from non-specific amplification product. The modified primers of the 
present invention are particularly useful in kinetic PCR because they not 
only reduce the amount of primer dimer formed, but also delay the 
formation of detectable amounts of primer dimer. A delay of primer dimer 
formation until after a significant increase in target sequence has 
occured enables independent monitoring of the amplification of target 
sequencs and minimizes the interference from primer dimer. 
Kits 
The present invention also relates to kits, multicontainer units comprising 
useful components for practicing the present method. A useful kit contains 
primers, at least one of which is modified as described herein, for 
nucleic acid amplification. Other optional components of the kit include, 
for example, an agent to catalyze the synthesis of primer extension 
products, the substrate nucleoside triphosphates, appropriate reaction 
buffers, and instructions for carrying out the present method. 
The examples of the present invention presented below are provided only for 
illustrative purposes and not to limit the scope of the invention. 
Numerous embodiments of the invention within the scope of the claims that 
follow the examples will be apparent to those of ordinary skill in the art 
from reading the foregoing text and following examples. 
EXAMPLE 1 
Synthesis of Primers Modified with a Benzyl Group 
Primers modified by the addition of the benzyl group were synthesized by 
one of two processes, described below. Primers modifed at the 3' terminal 
base were synthesized using N.sup.6 -benzyldeoxyadenosine Controlled Pore 
Glass (CPG) to initiate the DNA synthesis. Primers modified at an internal 
base were synthesized using an N.sup.6 -benzyldeoxyadenosine 
phosphoramidite. 
The following standard abbreviations are used in the example: 
______________________________________ 
DMAP 4-Dimethylaminopyridine 
DMF N,N-Dimethylformamide 
TEA Triethylamine 
EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, 
hydrochloride 
THF Tetrahydrofuran 
DMT 4,4'-Dimethoxytrityl 
LCAA-CPG Long Chain Alkyl Amino controlled pore glass 
______________________________________ 
I. Synthesis of N.sup.6 -benzyldeoxyadenosine CPG 
Step 1: Synthesis of N.sup.6 -benzoyl, N.sup.6 -benzyl, 
5'-O-DMT-2'-deoxyadenosine 
To N.sup.6 -Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (657 mg, 
1.0 mmol; Aldrich Chemical Co., Milwaukee, Wisc.), pyridine (10 ml) was 
added and the mixture was dried by evaporation under vacuum. This was 
repeated. The resulting foam was dissolved in anhydrous DMF (15 ml; 
Aldrich Chemical Co., Milwaukee, Wisc.) and cooled to 5.degree. C. Sodium 
hydride (44 mg, 1.1 mmol, 1.1 equiv. 60% dispersion in oil) was added 
under an argon atmosphere and stirred at room temperature for 45 minutes. 
Benzyl bromide (143 .mu.l, 206 mg, 1.2 mmol, 1.2 equiv; Aldrich Chemical 
Co., Milwaukee, Wisc.) was added over 2 minutes and the mixture was 
stirred overnight at room temperature. The mixture was dried by 
evaporation under vacuum and the residue was partitioned between ethyl 
acetate and water (10 ml each) and extracted. The aqueous phase was 
re-extracted with ethyl acetate (10 ml) and the combined extracts were 
dried over anhydrous magnesium sulfate, filtered and evaporated. The crude 
product was purified by column chromatography on silica gel (75 g) using 
methanol, triethylamine, methylene chloride (3:0.5:96.5). Fractions 
containing the product were combined and dried by evaporation to give the 
expected N.sup.6 -benzoyl, N.sup.6 -benzyl, 5'-O-DMT-2'-deoxyadenosine 
(410 mg, 54%). The structure of the product was confirmed by NMR. 
Step 2: Succinylation 
To the N.sup.6 -benzoyl, N.sup.6 -benzyl, 5'-O-DMT-2'-deoxyadenosine (295 
mg, 0.39 mmol), pyridine (10 ml) was added and the mixture was dried by 
evaporation under high vacuum. This step was repeated. Fresh anhydrous 
pyridine (10 ml) was added together with succinic anhydride (200 mg, 2 
mmol, 5.0 equiv) and DMAP (24 mg), and the solution was stirred under an 
argon atmosphere overnight at room temperature. The bulk of the solvent 
was removed under vacuum and the residue was partitioned between methylene 
chloride (20 ml) and sodium citrate solution (20 ml, 0.1 M, pH 5.0) and 
extracted. The aqueous phase was extracted with more methylene chloride 
(20 ml) and the combined extracts were dried over anhydrous sodium 
sulfate, filtered, and dried by evaporation. The product was purifed by 
column chromatography on silica gel (4.5 g) using ethyl acetate, 
triethylamine, methylene chloride (32:1:67) to give the expected 
3'-succinate ester, N.sup.6 -benzoyl-N.sup.6 
-benzyl-3'-O-succinate-5'-O-DMT-2'-deoxyadenosine (247 mg, 74%). 
Step 3: Derivatization of CPG 
Acid washed CPG was prepared a follows. LCAA-CPG (1.0 g, LCA00500C, 500 
angstrom, 88.6 .mu.mol/g; CPG Inc., Fairfield, N.J.) was washed with 
dichloroacetic acid in dichloromethane (2%, 20 ml) by swirling 
periodically over 20 minutes at room temperature. The acid washed CPG was 
filtered on a glass frit and washed with dichloromethane until acid free. 
The powder was air dried, then dried under vacuum at room temperature 
overnight. 
Coupling of the modified nucleoside intermediate to the acid washed CPG was 
carried out as follows. To a solution of N.sup.6 -Benzoyl-N.sup.6 
-benzyl-3'-O-succinate-5'-O-DMT-2'-deoxyadenosine (170 mg, 0.2 mmol), 
prepared as described above, in dichloromethane (10 ml) was added TEA (100 
.mu.L), and the solution was concentrated to approximately 5 ml under an 
argon atmosphere. DMAP (12 mg, 0.1 mmol, 0.5 equiv), TEA (100 .mu.L), EDC 
(384 mg, 2.0 mmol, 10 equiv), and the acid-washed CPG from above were 
added in sequence. Anhydrous pyridine (5 ml) was added and the mixture was 
sealed and shaken for 3 days at room temperature. The CPG was filtered off 
under vacuum and washed extensively with isopropanol, then with 
dichloromethane, air dried, then dried under vacuum for 1 hour. 
Capping of the derivatized CPG was carried out as follows. To the dry 
derivatized CPG were added Cap A and Cap B solutions (5 ml each, Acetic 
anhydride/2,6-Lutidine/THF and 10% N-Methylimidazole in THF; Glen Research 
DNA synthesis reagents, Sterling, Va.) and the mixture was shaken for 4 
hours at room temperature. The CPG was filtered off under vacuum and 
washed extensively with isopropanol, then dichloromethane, air dried, then 
dried under vacuum overnight. 
II. Synthesis of N.sup.6 -Benzyl Deoxyadenosine Phosphoramidite. 
N.sup.6 -benzoyl, N.sup.6 -benzyl, 5'-O-DMT-2'-deoxyadenosine was 
synthesized as described above. 
To N 6-benzoyl, N6-benzyl, 5'-O-DMT-2'-deoxyadenosine (196 mg, 0.26 mmol) 
in dry THF (8 ml) was added diisopropylethylamine (350 .mu.L, 270 mg, 2.04 
mmol, 7.8 equiv) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite 
(161 mg, 0.68 mmol, 2.6 equiv.; Aldrich Chemical Co., Milwaukee, Wisc.), 
and the mixture was stirred for 30 minutes at room temperature under an 
argon atmosphere. The solvent was removed under vacuum and the residue was 
partitioned between sodium bicarbonate solution (5%, 20 ml) and ethyl 
acetate (20 ml). The organic phase was washed with the bicarbonate 
solution, water, and saturated brine (20 ml each) in sequence, dried over 
sodium sulfate, filtered, and evaporated. The residue was purified by 
column chromatography on silica gel (4 g) using acetone/hexane/TEA 
(34:65:0.7) to yield the desired phosphoramidite (248 mg, 100%). 
III. DNA Synthesis purification and analysis. 
The benzyl derivatized adenosine CPG (25 mg, 1.0 .mu.mol) was transferred 
into empty synthesis columns (Glen Research, Sterling, Va.) and these were 
used to make oligonucleotides on an ABI 374 DNA synthesizer (Perkin Elmer, 
Applied Biosystems Division, Foster City, Calif.) using conventional 
synthesis and deprotection conditions. The crude DMT-DNA was purified and 
converted to the 5'-hydroxy-DNA by standard DMT On/Off HPLC using a Rainin 
Pure-DNA column on a Rainin HPLC system (Rainin Instrument Co, Woburn, 
Mass.). The oligonucleotides were analyzed using a ABI capillary 
electrophoresis system (Perkin Elmer, Applied Biosystems Division, Foster 
City, Calif.) or by denaturing anion-exchange HPLC chromatography on a 
Dionex Nucleopak column (Dionex Corp, Sunnyvale, Calif.). 
Similarly, synthesis of internally-modified primers was carried out using 
an unmodified CPG and the modified phosphoramidite synthesized as above. 
EXAMPLE 2 
Synthesis of Primers Modified with a t-Butyl-benzyl Group 
The present example describes the synthesis of primers modified at the 3' 
terminal adenosine with a p-tert-butylbenzyl group. The modified primers 
were synthesized essentially as described in Example 1, but using a 
N.sup.6 -(p-tert-Butylbenzyl)deoxyadenosine CPG. The synthesis of the 
derivatized CPG is described below. 
Step 1: Synthesis of N.sup.6 -benzoyl-N.sup.6 
-(p-tert-butylbenzyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine 
To N.sup.6 -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (658 mg, 
1.0 mmole) was added DMF (anhydrous, 10 ml) and evaporated to dryness. 
This was repeated. Fresh DMF (10 ml) was added under an Argon atmosphere. 
Sodium hydride (44 mg, 60% in oil, 1.1 mmole) was added and the mixture 
was stirred for 0.5 hour at room temperature. 4-(tert-butyl)benzyl bromide 
(272 mg, 1.2 mmole) was added dropwise and stirred at room temperature 
overnight. The solvent was removed under vacuum, and the residue was 
partitioned between ethyl acetate and water (20 ml each). The organic 
phase was washed with water (3 times, 20 ml), dried over anhydrous 
magnesium sulfate, filtered and evaporated to dryness. The crude product 
was purified by column chromatography on silica gel (100 g), using 
methylene chloride:methanol:triethylamine 96.5:3:0.5 to yield N -benzoyl-N 
-(p-tert-butylbenzyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, (229 
mg, 28.5%). 
Step 2: Succinylation. 
N.sup.6 -benzoyl-N.sup.6 -p-tert-butylbenzyl)-5'-O-(4,4' 
-dimethoxytrityl)-2'-deoxyadenosine (217 mg, 0.27 mmol) w as treated with 
succinic anhydride (135 mg, 5 equiv)and DMAP (17 mg, 0.5 equiv) in 
pyridine (10 ml). Work-up and chromatography as described in Example 1, 
above, yielded N.sup.6 -benzoyl-N.sup.6 -(p-tert-butylbenzyl)-5'-O-(4,4' 
-dimethoxytrityl)-2'-deoxyadenosine, 3'-O-succinate (199 mg, 82%). 
Step 3: Derivatization of CPG 
The succinate (180 mg, 0.2 mmol) from step 2, above, was treated with the 
acid washed LCAA-CPG as described in Example 1. The CPG was capped and 
vacuum dried to yield the N.sup.6 -benzoyl-N.sup.6 
-(p-tert-butylbenzyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 
3'-O-succinate derivatized CPG, (1.065 g). 
EXAMPLE 3 
Synthesis of Primers Modified with a Methyl Group 
Primers modified at the 3' terminal adenosine with a methyl group were 
synthesized using a N.sup.6 -methyl dA CPG (22 mg, 1 .mu.mole, Glen 
Research, Sterling Va.). The N.sup.6 -methyl dA CPG was placed in an empty 
synthesis column, and primers were made according to standard conditions 
of synthesis and deprotection. The primers were purified using the DMT 
On/Off HPLC procedure as described in Example 1. 
EXAMPLE 4 
Synthesis of Photo-Labile Modified Primers 
The present example describes the synthesis of primers modified at the 3' 
terminal adenosine with either one or two nitrobenzyl groups. The modified 
primers were synthesized essentially as described in Example 1, but using 
either a mononitrobenzyl dA CPG or a bis-nitrobenzyl dA CPG. 
I. Mononitrobenzalated primers 
The general method for the synthesis of N.sup.6 -benzoyl-N.sup.6 
-benzyl-2'-deoxyadenosine derivatized CPG (see Example 1) was applied to 
the synthesis of N.sup.6 -benzoyl-N.sup.6 
-ortho-nitrobenzyl-2'-deoxyadenosine derivatized CPG, by the substitution 
of ortho-nitrobenzylbromide as the alkylating agent. Subsequent steps for 
the CPG were identical to those described in Example 1, with the addition 
that the intermediates were protected from ambient light by wrapping the 
reaction flasks in aluminum foil. 
Following synthesis of the derivatized CPG, the primers were synthesized as 
described in Example 1, but were isolated by solid phase extraction using 
Nensorb Prep disposable columns (NEN Research Products Biotechnology 
Systems, Du Pont Co, Boston Mass.), using protocols as descibed by the 
manufacturer. 
II. Bis-nitrobenzylated primers 
Bis-nitrobenzyl deoxyadenosine CPG was synthesized as described below. 
Following synthesis of the derivatized CPG, the primers were synthesized 
and purified as described for the mononitrobenzyl primers. 
Step 1: Synthesis of 5'-O-DMT- N.sup.6 
-bis-ortho-nitrobenzyl-2'-deoxyadenosine. 
2'-Deoxyadenosine monohydrate (538 mg, 2.0 mmol, Aldrich Chemical, 
Milwaukee, Wisc.) was dried by evaporation with anhydrous pyridine (2 
times, 10 ml) under vacuum. The residue was dissolved in anhydrous DMF (10 
ml, Aldrich, Milwaukee, Wisc.) under an argon atmosphere, and sodium 
hydride (88 mg, 2.2 mmol, 1.1 equiv, 60% dispersion in oil) was added and 
stirred for 40 mins at room temperature. 2-Nitrobenzyl bromide (710 mg, 
3.3 mmol, 1.5 equiv) was added and the solution was stirred for 4 hours at 
room temperature. The DMF was removed by evaporation under vacuum, and the 
residue was partitioned between ethyl acetate and water (20 ml each). The 
aqueous phase was extracted with ethyl acetate (20 ml) and the combined 
extracts were washed with water (20 ml) and dried over magnesium sulfate, 
filtered and evaporated. The crude product was purified by column 
chromatography on silica gel (50 g, using 3% MeOH in CH.sub.2 Cl.sub.2) to 
yield 2'-deoxy-N.sup.6 -bis-ortho-nitrobenzyladenosine (320 mg, 30%). 
To 2'-deoxy-N -bis-ortho-nitrobenzyladenosine (200 mg, 0.518 mmol) was 
added anhydrous pyridine (10 ml) and evaporated to dryness. Pyridine (10 
ml) was added followed by 4-4'-dimethoxytrityl chloride (900 mg, 2.3 mmol, 
4.5 equiv.) and triethylamine (280 mg, 2.76 mmol, 4.0 equiv.) and stirred 
at room temperature under an argon atmosphere for 5 hours. Water (0.5 ml) 
was added and stirred for 20 minutes. The mixture was partitioned between 
ether and water (20 ml each) and the aqueous phase was re-extracted with 
ether (20 ml). The extracts were combined and washed with water (20 ml) 
and dried over anhydrous sodium sulfate, filtered and evaporated. The 
material was purified by chromatography on silica gel (4 g, using 0.7-2.5% 
methanol in methylene chloride) to yield 5'-O-DMT- N.sup.6 
-bis-ortho-nitrobenzyl-2'-deoxyadenosine, (121 mg, 33%). 
Step 2: Succinylation 
5'-O-DMT- N.sup.6 -bis-ortho-nitrobenzyl-2'-deoxyadenosine (121 mg, 0.145 
mmol) was dried by evaporation with anhydrous pyridine (2 times, 3 ml). 
Pyridine (3 ml), succinic anhydride (58 mg, 0.58 mmol, 4 equiv.) and DMAP 
(11 mg, catalytic) were added, and the solution was stirred at room 
temperature for 3 days. The solution was evaporated in vacuo, and the 
residue was partitioned between methylene chloride (10 ml) and sodium 
citrate buffer (0.1 M, pH 5.0, 10 ml). The organic phase was dried over 
anhydrous sodium sulfate, filtered and evaporated to dryness. The crude 
product was purified by chromatography on silica gel (2 g, using EtOAc: 
CH.sub.2 Cl.sub.2 :TEA, 32:67:1 10 ml, then MeOH:CH.sub.2 Cl.sub.2, 3:97, 
25 ml) to yield a pale yellow foam, 5'-O-DMT-N.sup.6 
-bis-ortho-nitrobenzyl-2'-deoxyadenosine-3'-O-succinate, (138 mg, 99.5%). 
Step 3: Derivatization of the CPG 
Acid-washed LCAA-CPG was prepared as in Example 1. 
Coupling of the modified nucleoside intermediate to the acid wached CPG was 
carried out as follows. 5'-O-DMT- N 
-bis-ortho-nitrobenzyl-2'-deoxyadenosine-3'-O-succinate (37 mg, 0.04 mmol) 
was treated with TEA (16 .mu.l) in an amber colored glass vial, and 
evaporated. To this residue was added anhydrous pyridine (1.5 ml), TEA (2 
.mu.l), DMAP (2.4 mg), EDC (76 mg, 0.04 mmol) and acid-washed LCAA-CPG 
(200 mg), and the mixture was shaken on an orbital mixer for three days at 
room temperature. The CPG was filtered off under reduced pressure and 
washed extensively with isopropanol, then with methlene chloride, air 
dried, then dried under vacuum for 1 hour. 
Capping of the derivatized CPG was carried out as described in Example 1. 
EXAMPLE 5 
Amplifications using Modified Primers--Effect of Position of Modified 
Nucleotide 
To demonstrate the effect of the modified primers on the formation of 
primer dimer, comparisons were carried out of amplifications of HIV-1 RNA 
using both modified primers and unmodified primers. In addition, to assess 
the effect of the position of the modified nucleotide on the reduction of 
primer dimer, amplifications were carried out using three different 
upstream modified primers, which differed only in the location of the 
modified base. 
Target Nucleic Acid 
HIV-1 RNA templates were synthesized using an HIV-1 RNA transcription 
vector essentially as described in Mulder et al., 1994, J. Clin. 
Microbiol. 32(2):292-300. 
Primers 
Amplifications were carried out using both unmodified and modified primers. 
The nucleotide sequences of the unmodified primers are shown below, 
oriented in the 5' to 3' direction. Upstream primer RAR1032MB (SEQ ID NO: 
1) and downstream primer RAR1033MB (SEQ ID NO: 2) amplify a 175 base pair 
product corresponding to nucleotide positions 2956 to 3130 of the sequence 
of HIV-1 reference strain HXB2 (GenBank accession no. K03455). 
__________________________________________________________________________ 
Primer Seq. ID No. 
Sequence 
__________________________________________________________________________ 
RAR1032MB 
1 CAATGAGACACCAGGAATTAGATATCAGTACAA 
RAR1033MB 2 CCCTAAATCAGATCCTACATATAAGTCATCCA 
__________________________________________________________________________ 
The above primer designations refer to the unmodified primers. Unmodified 
primers were biotinylated at the 5' end. Modified primers were synthesized 
as described in Example 1, which consisted of the same nucleotide 
sequences as the unmodified primers, but containing a benzylated adenosine 
at either the 3' terminal position or at a position one or three 
nucleotides upstream of the 3' terminus. The modified forms of the primers 
are designated herein as follows: 
Modified HIV-1 Amplification Primers 
______________________________________ 
Primer Seq Id. No. 
Position of Modified Nucleotide 
______________________________________ 
RAR1032MBA1 
1 3' terminus 
RAR1032MBA2 1 1 from 3' terminus 
RAR1032MBA4 1 3 from 3' terminus 
RAR1033MBA1 2 3' terminus 
______________________________________ 
Amplification 
Amplifications were carried out in 100 .mu.l reactions containing the 
following reagents: 
100 copies of HIV template RNA 
50 mM Tricine (pH 8.33), 
110 mM KOAc, 
300 .mu.M each dATP, dCTP, and dGTP, 
50 .mu.M dTTP 
500 .mu.M dUTP, 
50 .mu.M of each primer, 
3.5 mM Mn(OAc).sub.2, 
13% Glycerol. 
20 units of Z05 DNA polymerase*, and 
2.0 units of UNG**. 
FNT * described in U.S. Pat. No. 5,455,170 
FNT * * manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
Amplification temperature cycling was carried out in a TC480 DNA thermal 
cycler (Perkin Elmer, Norwalk, Conn.) using the following temperature 
profile: 
______________________________________ 
Pre-reaction incubation 
45.degree. C. for 4 minutes; 
Reverse-transcription 60.degree. C. for 20 minutes; 
46 cycles: denature at 94.degree. C. for 45 seconds, 
anneal/extend at 60.degree. C. for 45 seconds; 
Final extension 60.degree. C. for 7 minutes; 
Post-reaction hold 10.degree. C. until analysis (for a short time). 
______________________________________ 
Detection of Amplified Product 
The presence of amplified product was detected by gel electrophoresis as 
follows. Reaction products were fractionated using an agarose gel (100 ml 
of 3% NuSieve and 0.5% SeaChem) and 1.times.TBE (0.089 M Tris, 0.089 M 
boric acid, 0.0025 M disodium EDTA) running buffer were used. Ethidium 
bromide (0.5 .mu.g/ml) was added to stain any DNA present. Electrophoresis 
was carried out at 100 volts for approximately 1 hour. The ethidium 
bromide-stained bands of DNA were visualized using UV irradiation. 
Results 
The results of the gel electrophoretic analysis are seen in FIG. 1. The 
lane numbers corresponding to each of the amplifications using 
combinations of the unmodified and modified primers are shown in the table 
below. The bands corresponding to the intended HIV product are indicated 
in the figure by an arrow. The other bands in the gel correspond to 
non-specific amplification product and, in particular, primer dimer. 
______________________________________ 
Primers 
Upstream Downstream Lane No. 
______________________________________ 
RAR1032MB RAR1033MB 1 
RAR1032MBA1 RAR1033MB 2 
RAR1032MBA2 RAR1033MB 3 
RAR1032MBA4 RAR1033MB 4 
RAR1032MB RAR1033MBA1 5 
RAR1032MBA1 RAR1033MBA1 6 
RAR1032MBA2 RAR1033MBA1 7 
RAR1032MBA4 RAR1033MBA1 8 
______________________________________ 
Because the formation of primer dimer competes with the formation of the 
intended amplification product, a reduction in primer-dimer typically 
results in a concomitant increase in the amount of intended product 
formed. Thus, the effect of the modified primers can be seen by comparing 
the amount of primer-dimer formed relative to the amount formed using 
unmodified primers and by comparing the amount of intended target formed 
relative to the amount formed using unmodified primers. 
A comparison of the results using two unmodified primers (lane 1) to the 
results using a single 3'-modified primer (lanes 2 and 5) and to the 
results using two 3'-modified primers (lane 6) indicates that a decrease 
in primer dimer was obtained using either one or two modified primers. In 
amplifications using a single 3'-modified primer, a small difference in 
the reduction of primer dimer was seen which depended on which primer was 
modified. The use of two modified primers (lane 6) resulted in both the 
greatest decrease in primer dimer and a detectable increase in the amount 
of amplified target sequence. 
The effect of the position of the modified nucleotide is seen in a 
comparison of lanes 6-8. The reduction of primer dimer obtained using a 
primer modified at the nucleotide adjacent to the 3' terminal nucleotide 
(lane 7) was equivalent to that obtained using a primer modified at the 3' 
terminal nucleotide (lane 6), whereas the improvement obtained using a 
primer modified at the nucleotide three bases upstream of the 3' terminal 
nucleotide (lane 8) was slightly less. 
EXAMPLE 6 
Further Amplifications using Modified Primers--Effect of Position of 
Modified Nucleotide 
To further demonstrate the effect of the modified primers on the formation 
of primer dimer, comparisons were carried out of amplifications of HCV RNA 
using both modified primers and unmodified primers, essentially as 
described above. Amplifications were carried out using three different 
modified downstream primers, which differed only in the location of the 
modified base. 
Target Nucleic Acid 
HCV RNA templates were synthesized using an HCV RNA transcription vector as 
described in Young et al., 1993, J. Clin. Microbiol. 31(4):882-886. 
Primers 
Amplifications were carried out using both unmodified and modified primers. 
The nucleotide sequences of the unmodified primers are shown below, 
oriented in the 5' to 3' direction. Upstream primer ST280A (SEQ ID NO: 3) 
and downstream primer ST778AA (SEQ ID NO: 4) amplify a 240 base pair 
product from the 5' untranslated region of the HCV genome. 
HCV Amplification Primers 
______________________________________ 
Primer Seq Id No: 
Nucleotide Sequence 
______________________________________ 
ST280A 3 GCAGAAAGCGTCTAGCCATGGCGTTA 
ST778AA 4 GCAAGCACCCTATCAGGCAGTACCACAA 
______________________________________ 
The above primer designations refer to the unmodified primers. Modified 
primers were synthesized as described in Example 1, which consisted of the 
same nucleotide sequences as the unmodified primers, but contained a 
benzylated adenosine at either the 3' terminal position or at a position 
one or three nucleotides upstream of the 3' terminus. The modified forms 
of the primers are designated herein as follows: 
Modified HCV Amplification Primers 
______________________________________ 
Primer Seq Id. No. 
Position of Modified Nucleotide 
______________________________________ 
ST280ABA1 3 3' terminus 
ST778AABA1 4 3' terminus 
ST778AABA2 4 1 from 3' terminus 
ST778AABA4 4 3 from 3' terminus 
______________________________________ 
Amplification and Analysis 
Amplifications were carried out essentially as described in Example 3, but 
using 100 copies of HCV RNA template. Gel analysis of the amplified 
product was carried out as described in Example 3. 
Results 
The results of the gel electrophoretic analysis are seen in FIG. 2. The 
lane numbers corresponding to each of the amplifications using 
combinations of the unmodified and modified primers are shown in the table 
below. The bands corresponding to the intended HCV product are indicated 
in the figure by an arrow. The other bands in the gel correspond to 
non-specific amplification product and, in particular, primer dimer. 
______________________________________ 
Primers 
Upstream Downstream Lane No. 
______________________________________ 
ST280A ST778AA 1 
ST280A ST778AABA1 2 
ST280A ST778AABA2 3 
ST280ABA ST778AABA4 4 
ST280ABA1 ST778AA 5 
ST280ABA1 ST778AABA1 6 
ST280ABA1 ST778AABA2 7 
ST280ABA1 ST778AABA4 8 
______________________________________ 
Because the formation of primer dimer competes with the formation of the 
intended amplification product, a reduction in primer-dimer typically 
results in a concomitant increase in the amount of intended product 
formed. Thus, the effect of the modified primers can be seen both by 
comparing the amount of primer-dimer formed relative to the amount formed 
using unmodified primers and by comparing the amount of intended target 
formed relative to the amount formed using unmodified primers. 
The results obtained were similar to those obtained from the HIV 
amplifications described in the previous example, but in the HCV 
amplifications, the increase in intended product was more apparent than in 
the HIV amplifications. A comparison of the results using two unmodified 
primers (lane 1) to the results using a single 3'-modified primer (lanes 2 
and 5) and to the results using two 3'-modified primers (lane 6) indicates 
that a decrease in primer dimer was obtained using either one or two 
modified primers. The use of two modified primers (lane 6) resulted in 
both the greatest decrease in primer dimer along with a significant 
increase in the amount of amplified target sequence. As in the previous 
example, a small difference in the reduction of primer dimer was seen in 
amplifications using a single 3'-modified primer that depended on which 
primer was modified. 
The effect of the position of the modified nucleotide is seen in a 
comparison of lanes 6-8. Essentially equivalent results were obtained 
using primers modified at the 3'-terminal nucleotide (lane 6), nucleotide 
adjacent to the 3'-terminal nucleotide (lane 7), and the nucleotide three 
bases upstream of the 3'-terminal nucleotide (lane 8). These results 
indicate that the modifier group can be attached to any of the four 
nucleotides at the 3' end of the primer. 
EXAMPLE 7 
Amplifications using Modified Primers--Effect of Modifier Group 
To further demonstrate the effect of the modified primers on the formation 
of primer dimer, and to demonstrate alternative primer modifications, 
comparisons were carried out of amplifications of HCV RNA using both 
modified primers and unmodified primers, wherein the primers were modified 
by the addition of one of three different modifier groups: benzyl, 
nitrobenzyl, and methyl groups. 
Amplification results were analyzed by two different methods. In one set of 
comparisons, the presence of primer-dimer was assayed by gel 
electrophoretic analysis of the reaction products. In a second set of 
comparisons, the formation of primer dimer was monitored during 
amplification using the kinetic PCR methods described above. 
Target Nucleic Acid 
HCV RNA templates were synthesized using an HCV RNA transcription vector as 
described in Young et al., 1993, J. Clin. Microbiol. 31(4):882-886. 
Amplification Primers 
Amplifications were carried out using both unmodified and modified primers. 
The modified primers consisted of the same nucleotide sequences as the 
unmodified primers, but were modified at the 3' terminal adenosine by the 
addition of a methyl group, an benzyl group, or a nitrobenzyl group. 
Primers were synthesized as described in the previous examples. The 
designations for the primers used are shown below. 
______________________________________ 
Primer Seq Id. No. 
Modification of 3' Base 
______________________________________ 
ST280A 3 unmodified 
ST280AMEA1 3 methyl 
ST280ABA1 3 benzyl 
ST280ANBA1 3 nitrobenzyl 
ST778AA 4 unmodified 
ST778AAMEA 4 methyl 
ST778AABA1 4 benzyl 
ST778AANBA1 4 nitrobenzyl 
______________________________________ 
Amplification Reactions 
Amplifications were carried out in 100 .mu.l reactions containing the 
following reagents: 
0, 20, or 200 copies HCV RNA template 
50 mM Tricine, pH 8.3; 
110 mM KOAc; 
3.5 mM Mn(OAc).sub.2 ; 
300 .mu.M each dATP, dCTP, dGTP; 
50 .mu.M dTTP; 
500 .mu.M dUTP; 
250 nM each primer; 
20 U rTth*; 
2U UNG*; and 
13% Glycerol. 
FNT * manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
Thermal cycling of each reaction mixture was carried out in a GeneAmp.RTM. 
PCR System 9600 thermal cycler (Perkin Elmer, Norwalk, Conn.) using the 
following temperature profile: 
______________________________________ 
Pre-reaction incubation 
45.degree. C. for 4 minutes; 
Reverse-transcription 60.degree. C. for 24 minutes; 
46 cycles: denature at 94.degree. C. for 30 seconds, 
anneal/extend at 60.degree. C. for 30 seconds; 
Final extension 60.degree. C. for 7 minutes 
Post-reaction hold 4.degree. C. 
______________________________________ 
Detection of Amplified Product 
A. Gel Electrophoresis 
The presence of amplified product was detected by gel electrophoresis as 
follows. Reaction products were fractionated using an agarose gel (100 ml 
of 3% NuSieve, 0.5% SeaChem, and 0.5 .mu.g/ml ethidium bromide) and 
1.times.TBE (0.089 M Tris, 0.089 M boric acid, 0.0025 M disodium EDTA) 
running buffer. Electrophoresis was carried out at 100 volts for 
approximately 1 hour. The ethidium bromide-stained bands of DNA were 
visualized using UV irradiation. 
B. Detection by Kinetic PCR 
In the kinetic PCR methods described above, a intercalating dye such as 
ethidium bromide, which fluoresces more strongly when intercalated into 
double-stranded DNA, is added to the PCR. The increase in double-stranded 
DNA during amplification is monitored by measuring the fluorescence of the 
dye during the reaction. Because the kinetic PCR methods only measure an 
increase in the total amount of double-stranded DNA, formation of 
non-specific amplification product is not measured independently. In order 
to measure the occurence of non-specific amplification resulting from 
primer-dimer independent of template amplification, reactions were carried 
out without template nucleic acid. In such template-free reactions, any 
increase in double-stranded DNA is attributable to the formation of 
template-independent, non-specific amplification product. 
Kinetic PCR reaction conditions were as described above, except that 
ethidium bromide was added to the reaction mixture at a concentration of 1 
Mg/ml. Reactions were monitored by measuring the fluorescence of the 
reaction mixture as described in copending U.S. patent application Ser. 
No. 08/266,061, incorporated herein by reference. 
Fluorescence measurements were normalized by dividing by an initial 
fluorescence measurement obtained during a cycle early in the reaction 
while the fluorescence measurements between cylces were relatively 
constant. The cycle number chosen for the initial fluorescence measurment 
was the same for all reactions compared, so that all measurements 
represent increases relative to the same reaction cycle. Reaction 
fluorescence in target-free reactions remained relatively constant until 
primer dimer formed. In most reactions, if enough amplification cycles are 
carried out, primer dimer eventually becomes detectable. The effect of the 
modified primers can be seen from a comparison of the number of cycles 
carried out until primer dimer is formed, if at all. 
Results 
The results of the gel electrophoretic analysis are seen in FIG. 3. The 
lane numbers corresponding to each of the amplifications using the 
unmodified and three types of modified primers and 200 copies, 20 copies, 
or 0 copies of HCV RNA are shown in the table below (lanes numbers are 
counted from left to right: lanes 1-30 are in the top half of the gel; 
lanes 31-60 are in the bottom half of the gel). In addition, molecular 
weight markers were present in lanes 1 and 31 (Hae III digested PhiX 174 
RF DNA, New England Bioloabs, Beverly, Mass.) and in lanes lanes 30 and 60 
(Superladder-low, 20 bp ladder, Gen Sura, Del Mar, Calif.). The bands 
corresponding to the intended specific product are indicated in the figure 
by an arrow (.about.230 bp). The other bands in the gel correspond to 
non-specific amplification product and, in particular, primer dimer. 
Lane Numbers of Amplifications Results Shown in FIG. 3 
______________________________________ 
Templates Primers Lanes 
______________________________________ 
200 unmodified 2-5 
200 methylated 6-9 
200 benzylated 10-13 
200 nitrobenzylated 14-17 
20 unmodified 18-21 
20 methylated 22-25 
20 benzylated 26-29 
20 nitrobenzylated 32-35 
0 unmodified 36-41 
0 methylated 42-47 
0 benzylated 48-53 
0 nitrobenzylated 54-59 
______________________________________ 
The results demonstrate that amplification using the modified primers 
resulted in a greater amount of amplified HCV nucleic acid than 
amplifications using the unmodified primers. In addition, amplification 
using the modified primers resulted in a reduction in primer dimer 
relative to amplifications using the unmodified primers. 
In the kinetic PCR assays, the fluorescence was monitored throughout the 
reaction. The rate of increase of fluorescence after the increase in 
fluorescence was detectable was approximately the same in all reactions, 
as evidenced by the shape of the curve obtained plotting fluorescence 
versus cycle number (not shown). This indicated that the modified primers 
do not detectably inhibit the efficiency of each amplification step after 
the initial stage of amplification. The reactions differed significantly 
in the number of cycles carried out before an increase in fluorescence was 
detectable. 
To quantify the differences among the reactions, the results are expressed 
in terms of the number of amplification cycles carried out until the 
fluorescence exceeded an arbitrary fluorescence level (AFL). The AFL was 
chosen close to the baseline fluorescence level, but above the range of 
random fluctuations in the measured fluorescence, so that the reaction 
kinetics were measured during the geometric growth phase of the 
amplification. Accumulation of amplified product in later cycles inhibits 
the reaction and eventually leads to a reaction plateau. 
The kinetic PCR results are summarised in the table below. Each value for 
amplifications of 20 or 200 copies of target template represents an 
average of five replicate amplifications, with the exception of 
amplifications using benzylated primers and 20 copies of target, which 
represent an average of four replicates. Each value for amplifications 
without template represents an average of eight replicates. 
Two out of the eight replicates of amplifications using benzylated primers 
with no target present did not result in primer dimer formation by the end 
of the 46 cycles. The average of the remaining six amplifications is 
shown, which represents an average conditioned on primer dimer being 
formed. The conditional average is not comparable to the other values 
shown because of the deleted data. 
Cycles to Reach AFL 
______________________________________ 
Target copy number 
Primer 0 20 200 
______________________________________ 
unmodified 35 36 34 
methyl 39 38 36 
nitrobenzyl 43 40 37 
benzyl (43*) 41 37 
______________________________________ 
*2/8 showed no primer dimer formation 
The data indicate that the modified primers apparently delay the 
amplification of target nucleic acid such that the AFL is reached several 
cycles later. The delay did not correspond to a reduction in the final 
yield of specific amplification product. All amplifications of target 
nucleic acid were observed to reach a plateau within the 46 cycles used in 
the experiment and, as evidenced by the corresponding data from the gel 
electrophoretic analysis, the final yield was increased using the modified 
primers. 
The data indicate that the delay in the formation of primer dimer was 
significantly greater than the delay in the amplification of target. The 
benificial effect of the primers is most clearly seen comparing 
target-free amplifications and amplifications of 200 copies of template. 
Using unmodified primers, the increase in fluorescence to the AFL occured 
only one cycle later in amplifications without target, which indicates 
that amplification of target would be difficult to distinguish from the 
formation of primer dimer. In contrast, using modified primers, the 
increase in fluorescence due to primer dimer occured at least three cycles 
later and, using the benzylated primers, occured at least 6 cycles later, 
if it occured at all. Thus, target amplification could be detected and 
distinguished from the formation of primer dimer. 
Comparing target-free amplifications and amplifications of 20 copies of 
template, the effect of the modified primers showed the same pattern of a 
greater delay in the onset of primer dimer than the delay in target 
amplification. Using unmodified primers, 20 copies of template could not 
be detected. Using the nitrobenzyl and benzyl primers, the formation of 
primer dimer was delayed sufficiently so as to enable the detection of 20 
copies of template in this system. 
The data from monitoring the fluorescence at each amplification cycle (data 
not shown), indicated that, in general, the delay in primer dimer 
formation was sufficient to prevent primer dimer formation from reaching a 
plateau level within the 46 cycles. Thus, the modified primers appeared to 
delay the formation of primer dimer sufficiently such that amplification 
of target can be completed and the reaction stopped before a significant 
level of primer dimer is formed. 
EXAMPLE 8 
Photo-Labile Primers 
To demonstrate the use of photo-labile modified primers, amplifications of 
HCV RNA were carried out using both modified primers and unmodified 
primers. The modified primers were modified by the attachment of one or 
two nitrobenzyl groups to the exocyclic amine of the 3' terminal adenine. 
Amplification Primers 
Primers were synthesized as described in Example 4. The designations for 
the primers used are shown below. 
______________________________________ 
Primer Seq Id. No. Modification of 3' Base 
______________________________________ 
ST280A 3 unmodified 
15239 3 bis-nitrobenzyl 
15241 3 mononitrobenzyl 
ST778AA 4 unmodified 
15240 4 bis-nitrobenzyl 
15242 4 mononitrobenzyl 
______________________________________ 
Amplification Reactions 
For each primer pair, reactions were carried out using a dilution series of 
input target concentration. Two panels of the reactions, each including 
all combinations of primer pair and input target concentration, were 
carried out, and within each reaction panel, each reaction containing a 
given primer pair and target concentration was carried out in duplicate. 
Amplifications were carried out in 100 .mu.l reactions containing the 
following reagents: 
0, 10, 10.sup.2, 10.sup.3, 10.sup.4 or 10.sup.5 copies HCV RNA template 
55 mM Tricine, 90 mM KOAc, 
3 mM Mn(OAc).sub.2, 
200 .mu.M each dATP, dCTP, dGTP, dTTP, 
200 .mu.M dUTP, 
250 nM each primer, 
10 U rTth*, 
2 U UNG*, and 
8% Glycerol. 
FNT * manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
Thermal cycling of each reaction mixture was carried out in a GeneAmp PCR 
System 9600 thermal cycler (Perkin Elmer, Norwalk, Conn.) using the 
following temperature profile: 
______________________________________ 
Pre-reaction incubation 
50.degree. C. for 5 minutes; 
Reverse-transcription 60.degree. C. for 30 minutes; 
Initial denaturation 95.degree. C. for 1 minute; 
2 cycles: denature at 95.degree. C. for 15 seconds, 
anneal/extend at 60.degree. C. for 20 seconds; 
46 cycles: denature at 90.degree. C. for 15 seconds, 
anneal/extend at 60.degree. C. for 20 seconds; 
Final extension 72.degree. C. for 10 minutes 
______________________________________ 
Polished reaction tube caps (Perkin Elmer, Norwalk, Conn.) were used 
throughout. After the reaction temperature was raised to 60.degree. C. for 
the reverse-transcription step, the heated lid was removed from the PCR 
tray in the block of the thermal cycler, and half of the reaction tubes 
(one complete set of the duplicate reactions) were covered with aluminum 
foil. The other half was illuminated using a hand-held UV lamp emitting at 
302 mn (UVP model UVM-57, UVP Products, San Gabriel, Calif.) for ten 
minutes. The heated cover was replaced and the amplification was 
continued. 
Results 
The results of the amplifications were analyzed by gel electrophoresis as 
described above. The results are seen in FIG. 4. The primers and template 
copy number used in each reaction are indicated in the gel (log of the 
copy number shown). The bands corresponding to the intended product are 
indicated in the figure. The other bands in the gel correspond to 
non-specific amplification product and, in particular, primer dimer. 
A comparison of the UV-irradiated set of reactions shows that the use of 
the modified primers resulted in a significant decrease in primer dimer, 
especially at low copy numbers. 
A comparison of the non-irradiated set of reactions shows that the use of 
the bis-nitrobenzyl primers resulted in a complete inhibition of the 
amplification, as expected. Amplifications using the mononitrobenzyl 
primers not only yielded product, but exhibited a significant decrease in 
primer dimer, which is consistant with the results obtained in the 
previous example. 
EXAMPLE 9 
Amplifications using p-tert-butylbenzyl-Modified Primers 
This example describes the amplification of HCV RNA using primers modified 
with p-tert-butylbenzyl groups. 
Target Nucleic Acid 
HCV RNA templates were synthesized using an HCV RNA transcription vector as 
described in Young et al., 1993, J. Clin. Microbiol. 31(4):882-886. 
Primers 
Amplifications were carried out using modified primers synthesized as 
described in Example 2, above. The nucleotide sequences of the unmodified 
primers are shown below, oriented in the 5' to 3' direction. The primers 
used were modified versions of upstream primer ST280A (SEQ ID NO: 3) and 
downstream primer ST778AA (SEQ ID NO: 4). The modified forms of the 
primers are designated herein as follows: 
Modified HCV Amplification Primers 
______________________________________ 
Primer Seq Id. No. 
Position of Modified Nucleotide 
______________________________________ 
ST280ATBU 3 3' terminus 
ST778AATBU 4 3' terminus 
______________________________________ 
Amplification and Analysis 
Amplifications were carried out in 100 .mu.l reactions containing the 
following reagents: 
20, 5, 2.5, 2, or 0 copies of HCV template RNA 
50 mM Tricine (pH 8.33), 
110 mM KOAc, 
300 .mu.M each dATP, dCTP, and dGTP, 
50 .mu.M dTTP 
500 .mu.M dUTP, 
50 nM of each primer, 
3.5 mM Mn(OAc).sub.2, 
13% Glycerol. 
20 units of rTth DNA polymerase, and 
8.0 units of UNG*. 
FNT * manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
Amplification temperature cycling was carried out in a TC480 DNA thermal 
cycler (Perkin Elmer, Norwalk, Conn.) using the following temperature 
profile: 
______________________________________ 
Pre-reaction incubation 
45.degree. C. for 12 minutes; 
UNG inactivation 90.degree. C. for 30 seconds; 
Reverse-transcription 60.degree. C. for 20 minutes; 
47 cycles: denature at 94.degree. C. for 45 seconds, 
anneal/extend at 60.degree. C. for 70 seconds; 
Final extension 60.degree. C. for 7 minutes; 
Post-reaction hold 10.degree. C. until analysis (for a short time). 
______________________________________ 
The amplification products were analyzed by gel electrophoresis, as 
described above. 
Results 
Amplifications carried out at each target template number were replicated 
as follows: 3 amplifications were carried out using 20 copies of target 
template, 3 amplifications were carried out using 5 copies of target 
template, 2 amplifications were carried out using 2.5 copies of target 
template, 1 amplification was carried out using 2 copies of target 
template, and 23 amplifications were carried out with no target present. 
All template positive amplifications resulted in a single band on the gel 
of the expected target size. None of the amplifications resulted in either 
primer dimer or other non-specific amplification product. 
The results can be compared to those in Example 6, above, wherein the same 
HCV target was amplified using the same primer sequences. A comparison of 
these results to those in Example 6 indicate that amplifications using 
p-tert-butylbenzyl-modified primers were significantly improved relative 
to the corresponding amplifications carried out with unmodified primers. 
Additional experiments were carried out in which HIV-1 RNA was amplified 
using p-tert-butylbenzyl-modified versions of the primers described in 
Example 5, above. The amplifications were carried out essentially as 
described above. As with the HCV system described herein, all HIV-1 
template positive amplifications resulted in a single band on the gel of 
the expected target size, and none of the amplifications resulted in 
either primer dimer or other non-specific amplification product. 
These additional results can be compared to those in Example 5, above, 
wherein the same HIV target was amplified using the same primer sequences. 
A comparison of these results to those in Example 5, above, indicates that 
amplifications using p-tert-butylbenzyl-modified primers were 
significantly improved relative to the corresponding amplifications 
carried out with unmodified primers. 
EXAMPLE 10 
Amplification of Mycobacterial DNA 
This example describes a comparison of amplifications of mycobacterial DNA 
carried out using unmodified and modified primers. Both primers modified 
by the addition of a benzyl group to the 3' terminal nucleotice and 
primers modified by the addition of a p-tert-butylbenzyl group to the 3' 
terminal nucleotide were used. The reactions using unmodified primers were 
essentially as described in Tevere et al., 1996, J. Clin. Microbiol. 
34(4):918-923. Amplifications were carried out using sputum samples into 
which mycobacterial DNA had been added in a known concentration to mimic 
infected clinical samples. Additional amplifictions were carried out using 
purified mycobacterial DNA, and using DNA-free negative control samples. 
Sample Preparation 
Sputum specimens previously shown to be negative for mycobacteria by 
microscopy and culture were liquefied and decontaminated by the 
N-acetyl-cysteine-NaOH method recommended by the CDC (Kent and Kubica, 
1985, Public Health Mycobacteriology--a guide for the level III 
laboratory, U.S. Department of Health and Human Services, Centers for 
Disease Control, Atlanta, incorporated herein by reference). Liquefied 
sputum (100 .mu.l) was added to 500 .mu.l of Respiratory Specimen Wash 
Reagent (10 mM Tris-HCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.05% 
NaN.sub.3, pH8.0) and centrifuged for 10 minutes at 12,500.times.g. Each 
pellet was resuspended in 100 .mu.l of lysis reagent (0.05 N NaOH, 1% 
(v/v) Triton X-100, 1 mM EDTA, 0.05% NaN.sub.3) and incubated for 45 
minutes at 60.degree. C. The lysates were then neutralized with 100 .mu.l 
of neutralization reagent (0.2 M Tris-HCl, 8 mM MgCl.sub.2, 0.05% 
NaN.sub.3, pH 7.5). 
Pooled sputum lysates were generated by combining 80 .mu.l each of two 
separate sputum lysates. To each of 8 pooled sputum lysates (160 .mu.l 
each) were added 15 .mu.l of a DNA stock (2 copies/.mu.l in a 1:1 mixture 
of lysis and neutralization reagents) purified from cultured M 
tuberculosis. 
Samples containing purified mycobacterial DNA (no sputum) in a known 
concentration were prepared by adding 10 .mu.l of the DNA stock to 100 
.mu.l of a 1:1 mixture of lysis reagent and neutralization reagent. 
Negative control samples (no DNA) consisted a mixture of 100 .mu.l of lysis 
reagent and 100 .mu.l of neutralization reagent. 
Amplification Primers 
Amplifications were carried out using primers consisting of the following 
nucleotide sequences: 
______________________________________ 
Primers Sequence 
______________________________________ 
KY18 (SEQ ID NO:5) 
5'-CACATGCAAGTCGAACGGAAAGG-3' 
- KY436 (SEQ ID NO:6) 5'-TAACACATGCAAGTCGAACGGAAA-'3' 
- KY75 (SEQ ID NO:7) 5'-GCCCGTATCGCCCGCACGCTCACA-3' 
______________________________________ 
The following primer pairs, containing the indicated modifier group 
attached to the 3' terminal base, were used in the amplifications. All 
modified primers were synthesized as described in the previous examples. 
All primers were biotinylated at the 5' end. 
______________________________________ 
Primer Pair 
Primer Sequences 
Modification 
______________________________________ 
A KY18 (SEQ ID NO: 5) 
unmodified 
KY75 (SEQ ID NO: 7) unmodified 
B KY436 (SEQ ID NO: 6) benzyl 
KY75 (SEQ ID NO: 7) benzyl 
C KY436 (SEQ ID NO: 6) p-tert-butylbenzyl 
KY75 (SEQ ID NO: 7) p-tert-butylbenzyl 
______________________________________ 
Amplification 
For each sample, amplifications were carried out using the unmodified 
primer pair, KY18 (SEQ ID NO: 5) and KY75 (SEQ ID NO: 7), and modified 
forms of the primer pair, KY436 (SEQ ID NO: 6) and KY75 (SEQ ID NO: 7). 
Amplifications were carried out in 100 .mu.l reactions, each containing 50 
.mu.l of one of the three samples described above and 50 .mu.l of a 
2.times.reagent mixture, which contains the following reagents: 
100 mM Tris-HCl, pH 8.9; 
500 nM each primer; 
200 .mu.M (each) dNTP (dATP, dCTP, dGTP, dUTP); 
20% (v/v) glycerol; 
10 units AmpliTaq.RTM.*; 
6 units AmpErase.RTM.* 
FNT * Manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer (Norwalk, Conn.; 
Thermal cycling of each reaction was carried out in a GeneAmp PCR system 
9600 thermal cycler (Perkin Elmer, Norwalk, Conn.) using the following 
temperature profile: 
______________________________________ 
Pre-reaction incubation 
50.degree. C. for 5 minutes; 
2 cycles: denature at 98.degree. C. for 20 seconds, 
anneal at 62.degree. C. for 20 seconds, 
extend at 72.degree. C. for 45 seconds; 
41 cycles: denature at 94.degree. C. for 20 seconds, 
anneal at 62.degree. C. for 20 seconds, 
extend at 72.degree. C. for 45 seconds; 
Final extension 72.degree. C. for approximately 12 hours (overnight). 
______________________________________ 
Amplification products were visualized by electrophoresis through a 2% 
Nusieve.RTM., 0.5% agarose gel followed by ethidium bromide staining. 
Results 
The results of the electrophoretic analysis are shown in FIG. 5. For each 
sample, the products from amplifications carried out with unmodified 
primers (indicated "A") and with modified primers (indicated "B" and "C") 
were run on adjacent lanes. The bands corresponding to the intended 
mycobacterial target sequence are indicated with arrows. Other bands 
correspond to non-specific amplification product; the lowest bands in the 
gel correspond to primer dimer. Lanes marked "M" contain a molecular 
weight marker (Hae III digestion of PhiX174 DNA). 
Using the unmodified primers, amplifications of purified mycobacterial DNA 
resulted in the formation of primer dimer. The use of the either modified 
primer pairs increased the amount of intended target present and 
essentially eliminated the formation of detectable primer dimer. 
In contrast to amplifications of purified DNA, using the unmodified 
primers, the presence of sputum lysate in the amplification reaction 
reduced the efficiency and increased the formation of non-specific 
amplification product, as shown by the presence of extraneous product 
bands. The increase of non-specific amplification product is not 
surprising given that sputum lysates contain a significant amount of human 
DNA, which was not present in the amplifications of purified mycobacterial 
DNA. The use of the either of the modified primer pairs in amplifications 
carried out in the presence of sputum resulted in both a significant 
increase in the amount of intended product generated and a reduction of 
non-specific amplification. 
EXAMPLE 11 
Additional Synthesis of Primers Modified with a Benzyl Group 
Primers modified by the addition of a benzyl group to a terminal cytosine 
were synthesized essentially as described in Example 1, but using an 
LCAA-CPG-linked N.sup.4 -acetyl, N.sup.4 -benzyl-5'-O-DMT-2'-deoxycytidine 
prepared as described below. 
Step 1: Synthesis of N.sup.4 -benzyl-2'-deoxycytidine 
To 2'-deoxycytidine hydrochloride (5.28g, 20 mmol, U.S. Biochemical Corp., 
Cleveland, Ohio) was added benzylamine (20 ml), and the mixture was heated 
at 150.degree. C. for 3 hours under an argon atmosphere. The solution was 
concentrated under vacuum to yield a viscous yellow oil, which was 
partitioned between water (100 ml) and ethyl acetate (100 ml). The aqueous 
phase was washed with ethyl acetate (100 ml) and separated. The aqueous 
phase was concentrated under vacuum to yield a yellow syrup (13 g), which 
was purified by silica gel column chromatography with 15:1 methylene 
chloride:methanol as eluant, to yield the desired product (5.8 g, 91.5%), 
as a colorless syrup. 
Step 2: Synthesis of N.sup.4 -acetyl, N.sup.4 -benzyl-2'-deoxycytidine 
N.sup.4 -benzyl-2'-deoxycytidine (2.5g, 7.9 mmol) was dissolved in 15 ml 
dry dimethylformamide (15 ml), acetic anhydride (8g, 79 mmol, 10 eq.) was 
added, and the mixture was stirred overnight at room temperature. The 
solvent and excess acetic anhydride were evaporated under vacuum. The 
product was purified by column chromatography with silica gel using 20:1 
methylene chloride:methanol as eluant, to yield the title compound (1.3 g, 
48%). The compound was highly hygroscopic and was stored desiccated at 
-20.degree. C. 
Step 3: Synthesis of N.sup.4 -acetyl, N.sup.4 -benzyl, 
5'-O-DMT-2'-deoxycytidine. 
N.sup.4 -acetyl, N.sup.4 -benzyl-2'-deoxycytidine (76 mg, 0.2 mmol) was 
dissolved in 1 ml dry pyridine, and DMT-Cl (122 mg, 0.2 mmol, 1.0 eq) was 
added. The reaction mixture was stirred for 3 hours. Analysis by TLC 
showed some starting material was left, so a further aliquot of DMT-Cl (61 
mg, 0.5 eq) was added and the resulting mixture was stirred for another 
hour, at which time analysis by TLC showed that the reaction was complete. 
The reaction was quenched with 15 ml brine solution and the aqueous phase 
was extracted with methylene chloride (3.times.15 ml). The combined 
organic layer was washed with brine (2.times.15 ml) and dried over 
anhydrous magnesium sulfate. The solvent was evaporated and the mixture 
was purified by silica gel chromatography using 50:1 methylene chloride: 
methanol, to yield N.sup.4 -acetyl, N.sup.4 -benzyl, 
5'-O-DMT-2'-deoxycytidine (96 mg, 65% yield). 
Step 4: Succinylation 
N.sup.4 -acetyl, N.sup.4 -benzyl, 5'-O-DMT-2'-deoxycytidine (96 mg, 0.13 
mmol) was dissolved in 2 ml dry pyridine. Succinic anhydride (100 mg, 1.0 
mmol) and dimethylaminopyridine (20 mg) were added, and the resulting 
mixture was stirred at room temperature for three days. The solvent was 
evaporated and the residue was co-evaporated with toluene (3.times.10 ml). 
Chloroform (50 ml) was added to dissolve the residue (sonication was used 
to help the dissolution). The chloroform layer was washed with brine 
(3.times.15 ml), and water (1.times.15 ml). The organic layer was dried 
with anhydrous magnesium sulfate. The solvent evaporated to give 108 mg 
pure N.sup.4 -acetyl, N.sup.4 -benzyl, 
5'-O-DMT-2'-deoxycytidine-3'-O-succinate (97% yield). 
Step 5: Preparation of LCAA-CPG linked 5'-O-DMT- N.sup.4 -acetyl, N.sup.4 
-benzyl-2'-deoxycytidine-3 '-O-succinate 
Activated CPG was prepared as follows. LCAA-CPG (1.0 g, LCA00500C, CPG 
Inc., Fairfield, N.J.) was treated with trichloroacetic acid in methylene 
chloride (3%, 10 ml) and was mixed by rotation on a rotary evaporator 
(rotovapor, Buchi, Flawil, Switzerland) (no vacuum) for 4 hours. The 
solvent was filtered off and the CPG was washed with 9:1 
triethylamine:ethyldiisopropylamine (3.times.5 ml), methylene chloride 
(3.times.10 ml), and ether (3.times.10 ml) consecutively, then dried under 
vacuum. 
Coupling of the modified nucleoside intermediate to the acid washed CPG was 
carried out as follows. To 1 gram activated LCAA-CPG was added N.sup.4 
-acetyl, N.sup.4 -benzyl, 5'-O-DMT-2'-deoxycytidine, 3'-O-succinate (108 
mg, 0.13 mmol), prepared as described above, dimethylaminopyridine (20 
mg), and 5 ml dry pyridine. The reaction mixture was rotated on a 
rotavapor (no vacuum) for three days. The supernatant was filtered off, 
and the coupled LCAA-CPG was washed sequentially with pyridine (3.times.5 
ml), methylene chloride (3.times.10 ml), and ether (3.times.10 ml), and 
then dried in vacuum. 
Capping of the LCAA-CPG linked with N.sup.4 -acetyl, N.sup.4 -benzyl, 
5'-O-DMT-2'-deoxycytidine-3'-O-succinate was carried out as follows. To 
the derivatized CPG was added Capping mix reagent A (THF/Lutidine/Ac.sub.2 
O 8:1: 1, Glen Research DNA synthesis reagents, Sterling, Va.) and B (10% 
N-methylimidazole in THF, Glen Research), and the reaction mixture was 
rotated on a rotavapor (no vacuum) overnight. The solution was filtered 
off, and the coupled LCAA-CPG was washed sequentially with pyridine 
(3.times.5 ml), methylene chloride (3.times.10 ml), THF (3.times.10 ml), 
and ether (3.times.10 ml), and then dried under vacuum. 
The coupling capacity of the derivatized LCAA-CPG was determined by 
treating 5 mg of the product with 3% trichloroacetic acid in methylene 
chloride, and the amount of the released dimethoxyltrityl carbonium ion 
was measured by UV spectroscopy. The amount of nucleoside derivative 
linked to LCAA-CPG was determined to be 19.5 .mu.mol/g. 
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# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 7 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1: 
- - CAATGAGACA CCAGGAATTA GATATCAGTA CAA - # - 
# 33 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2: 
- - CCCTAAATCA GATCCTACAT ATAAGTCATC CA - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3: 
- - GCAGAAAGCG TCTAGCCATG GCGTTA - # - # 
26 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
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- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:4: 
- - GCAAGCACCC TATCAGGCAG TACCACAA - # - # 
28 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:5: 
- - CACATGCAAG TCGAACGGAA AGG - # - # 
23 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
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(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:6: 
- - TAACACATGC AAGTCGAACG GAAA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:7: 
- - GCCCGTATCG CCCGCACGCT CACA - # - # 
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