Note: Descriptions are shown in the official language in which they were submitted.
2(~
NN-0233
~ITLE
PROCESS FOR RAPID NUCLEIC ACID DETECTION
BY INCORPORATING A REPORTER M~IE~Y INTO
AMPLIFIED TARGET NUCLEIC ACID ~OLLOWED
BY AFFINITY CAPTU~E
FIELD OF INVENTION
This invention relates to the detection of
nucleic acid séquences and more specifically to a
process of combining amplification of target nucleic
acid sequences with detection of a reporter group
specifically incorporated into the target sequence
followed by affinity capture.
~aCKGROUND OF THE INVEN~IQN
The development of practical nucleic acid
hybridization methods which can be used for detecting
nucleic acid sequences of interest has been limited by
several factors. These include lack of sensitivity,
complexity of procedure, and the desire to convert
from radiometric to nonradiometric detection methods.
A variety of methods have been investigated for the
purpose of increasing the sensitivity nonradiometric
procedures. In one general approach, improvements in
the total assay procedure have been e~amined, with
concomitant effects on the issues of cDmplexity and
nonradiometric detection. In another approach,
methods which increase the amount of nucleic acid to
be detected by such assays have been pursued.
U.S. Patent 4,358,535, issued to ~alkow,
describes a method of culturing cells to increase
their number and thus the amount of nucleic acid of
the organism suspected to be present, depositing the
sample onto fixed support, and then contacting the
sample with a labeled probe, followed by was~ing the
support and detecting the label. One drawback to this
20i298~
method is that without culturing the organism first,
- the assay does not have adequate sensitivity. Adding a
culture step, however, is time consuming and not
always successful. Maniatis et al., Molecular
S Clonin~: A Laboratorv Manual, Cold Spring Harbor
Laboratory, pp.390-401 (1982), describe a method in
which a nucleic acid of interest is amplified by
cloning it into an appropriate host system. Then,
when the host organism replicates in culture, the
nucleic acid of interest is also replicated. This
method also suffers from the requirement to perform a
culture step and thus provides for a procedure that
is time consuming and complicated.
An alternative approach tG increasing the
quantity of nucleic acids of organisms has been
described in U.S. patents 4,683,202 and 4,683,195.
These patents disclose "a process for amplification
and detection of any target nucleic acid sequence
contained in a nucleic acid or mixture thereof". This
process employs an in vitro cycling mechanism which
doubles the nucleic acid sequence to be amplified
after each cycle is complete. This is carried out by
separating the complementary strands of the nucleic
acid sequence to be amplified, contacting these
strands with excess oligonucleotide primers and
extending the primers by enzymatic treatment to form
primer extension products that are complementary to
the nucleic acid annealed with each primer. The
process is then repeated as many times as is
necessary. An advantage of this method is that it can
rapidly produce large quantities of a small portion of
the sequence of the nucleic acid of an organism ~f
interest. A disadvantage of this method is that the
detection of the nucleic acids produced, using a
direct assay method, is complicated in that the
2~:2~34
amplification process can produce nucleic acid
sequences which are not faithful copies of the
original nucleic acid which was to be copied. These
erroneous nucleic acid sequences can provide false
positives in the assay which increase the background
noise and thus decrease the sensitivity of the entire
method.
Numerous DNA probe assays have been described in
the past for the detection of nucleic acids of
interest. Falkow's method (above) first renders the
target nucleic acid single-stranded and then
immobilizes it onto a solid support. A labeled probe
which is complementary to the target nucleic acid is
then brought into contact with the solid support. Any
excess probe is washed away and the presence of the
label in the resulting hybrid is determined. A
disadvantage of this method is that it is time
consuming and cumbersome. The assay steps, i.e.,
hybridization and washing steps are carried out in a
sealed pouch which contains the membrane (solid
support) as well as the buffer solution.
Hill et al., WO 86/05815, describe a variation of
the above assay format employing nitrocellulose coated
magnetic particles to which the target DNA is affixed,
followed by direct hybridization with a biotinylated
probe and detection using a streptavidin-conjugated
reporter.
Dunn et al., Cell, Vol. 12, 23-36 (1977~,
describe a different hybridization format which
employs a two-step sandwich assay method employing
polynucleotlde probes in which the target nucleic acid
is mixed with a solution containing a first or capture
probe which has been affixed to a solid support.
After a period of time, the support ~s washed and a
second or reporter (labeled) probe, also complementary
4 2012984
to the target nucleic acid but not to the capture
probe, is added and allowed to hybridize with the
capture probe - target nucleic acid complex. After
washing to remove any unhybridized reporter probe, the
presence of the reporter probe, hybridized to the
target nucleic acid, is detected.
Ranki et al. U.S. Patent 4,563,419, disclose
~PA 0 154 505, W086/03782, and EPA 0 200 113. It is
to be recognized that all of these employ an assay
procedure in which the first or capture probe is
immobilized onto a solid support prior to
hybridization.
A further variation has been described in German
Preliminary Published Application 3,546,312 A1. This
method, like that described by Ranki et al., empl~ys a
capture probe and a reporter probe which hybridize to
distinct portions of the target nucleic acid. The
target nucleic acid is contacted in solution by the
two probes. The first, or capture probe, contains a
binding component, such as biotin, that is capable of
binding with a receptor component, such as
streptavidin, which has been affixed to a solid
support. After formation of the capture probe -
target nucleic acid - reporter probe complex, a
streptavidin-modified solid support is added. Any
unhybridized reporter probe is washed away followed by
the detection of the label incorporated into the
complex bound to the solid support. An advantage of
this technique over that disclosed by Ranki et al. is
that the hybridization, which takes place in solution,
is favored kinetically. Some disadvantages are that
the length of the target nucleic acid affects the
overall efficiency of the reaction which decreases
with increasing target nucleic acid length. Also,
sandwich nucleic acid probe assays, whether
5 zo~2~84
heterogeneous two-step or one-step, or utilizing
solution hybridization, are not as sensitive as the
direct assay method.
pISCLOSURE OF TH~ INVENTION
S The nucleic acid assay of this invention for the
detection and/or measurement of a preselected nucleic
acid sequence in a sample suspected of including a
nucleic acid containing said preselected sequence
comprises the steps of:
(A) rendering the target nucleic acid single-
stranded;
(B) amplifying at least one specific nucleic
acid sequence contained within the
preselected nucleic acid sequence in the
presence of at least one
deoxyribonucleotide triphosphate containing
a reporter moiety in an amount up to the
total replacement of the corresponding
dNTP, by
(1) treating the strands with two
oligonucleotide primers, for each
different specific sequence being
amplified, under conditions such that
for each different sequence being
amplified an extension product of each
primer is synthesized which is
complementary to each nucleic acid
strand, wherein said primers are
selected so as to be sufficiently
complementary to the d~fferent strands
of each specific sequence to hybridize
therewith such that the extension
product synthesized from one primer,
when it is separated from it8
complement, can serve as a template
2~3~2~34
for synthesis of the extension pro~uct
of the other primer;
(2) separating the primer extension
products from the templates on which
they were synthesized to produce
single-stranded molecules; and
(3) treating the single-s~randed molecules
generated from step ~2) with the
primers of step ~1~ under conditions
that a primer extension product is
synthesized using each of the single
strands produced in step ~2) as a
template;
(4) repeating steps ~) and ~3) to produce
sufficient primer extension product
for detection and/or measurement:;
~C) contacting the product of step ~B) with a
solid affinity support matrix;
~D) removing any unincorporated reporter moiety; and
~E) detecting and/or measuring the reporter
moiety immobilized on the affinity support.
~AILED ~E~RIPTION OF THE INVENTION
The nucleic acid assay of this invention
comprises the following overall process for the
detection of target nucleic acids of a preselected
sequence:
a) Using the polymerase chain reaction ~PCR)
nucleic acid amplification method described in U.S.
4,683,202, incorporated herein by reference, specific
nucleic acid sequences are amplified by annealing the
denatured target nucleic acid present in the sample
with primers specific for the target and forming
extension products. In this process, a
deoxyribonucleotide triphosphate containing a reporter
group ~moiety), dNTP-R, is used to replace some or all
20~.2~84
of at least one of the corresponding
deoxyribonucleotide triphosphates employed. Each
extension product formed is complementary to a portion
of the preselected nucleic acid sequence contained
within the target nucleic acid and becomes a template
for further primer binding. This process ls then
repeated as necessary in order to produce the desired
amount of primer extension product for detection
and/or measurement.
b) The resulting nucleic acid is brought in
contact with an affinity support and allowed to bind
to the affinity support for a period of from 1 to 30
minutes. The affinity support is then rinsed with an
appropriate buffer in order to remove non-incorporated
reporter group modified deoxyribonucleotide
triphosphate.
The term "PCR" as used herein in referring to the
process of amplifying target nucleic acid sequences
employing primer oligonucleotides to produce by
enzymatic means a greatly increased number of copies
of a small portion of the target nucleic acid is
described in U.S. patent 4,683,202.
The PCR target amplification reaction requires
approximately 20 to 30 repeat cycles in order to
produce a sufficient quantity of the amplified target
nucleic acid for further hybridization. Denaturation
of the amplified nucleic acid can be accomplished by
treatment with alkali, acid, chaotropic agents, or
heat, although the preferred method is to place the
amplified target nucleic acid in a boiling water bath
for at least 10 minutes followed by a chilled water
bath ~4C) for at least two mlnutes.
A variety of affinity supports can be utilized.
Among these are known affinity membranes 9uch as Gene
Screen~ hybridization transfer membrane ~a registered
8 20~,2984
trademark of E.I. du Pont de Nemours and Company; a
nylon-based membrane), nitrocellulose, and Gene Screen
Plus~M, a positively charged and supported nylon 66
membrane. The method of transferring nucleic acids is
described in U.S. Patent 4,455,370 which is hereby
incorporated by reference.
A variety of known detection methods can be
utilized in the assay of this invention, depending
upon the reporter group incorporated into the
amplified product. When the reporter group is a
chromophor or fluorophor, then the incorporated
reporter group can be detected by known spectroscopic
techniques.
In an alternative detection method, a labeled
antibody to the reporter group incorporated during the
amplification process is employed. It is brought into
contact with the amplified product before or
subsequent to capture of the amplified product. The
label on the antibody can then be used to detect the
presence of the amplified product.
The Example below exemplifies the invention.
~XAMPLE
Detection of HIV I
A. Amplification of Target Nucleic
25Acid by PCR
The procedure as described in U.S. Patent
4,683,202 and in a product bulletin for GeneAmp DNA
Amplification Reagent ~it (~N801-0043) can be followed
utilizing the following specific conditions and
reagents. A 103-nucleotide base sequence located
within the GAG pl7 region of HIV I, incorporated lnto
a plasmid (the plasmid incorporating most of the HIV I
genome is designated pBH10-R3), can be amplified using
primers A and ~ as shown below:
9 ~n~ss4
5'-TGGGCAAGCAGGGAGCTAGG
Primer A
5'-TCTGAAGGGATGGTTGTAGC
Primer B
Aliquots of serial dilutions (lx10+7, lx10+6,
lx10+5, lx10+4, lx10+3, lx10+2, lxlO+1, and zero
copies) of plasmid p~HlO-R3 can be amplified using
PCR. Each aliquot can be combined with a buffer 200
~M in each of dATP, dTTP, dCTP, and dGTP, and 10 ~M in
succinyl-fluorescein dTTP, 1.0 ~M in each of Primer A
and Primer B, and containing l ~g of human placental
DNA/reaction and 2.5 units of a DNA polymerase enzyme,
in a total reaction volume of 100 ~l.
Each reaction mixture can then be temperature
cycled as described in the product bulletin thirty
(30) times.
This process is expected to result in the
estimated increase in the number of target molecules
by lx10+5 to lx10+6.
B. Capture
The product of Step A car. be adsorbed onto a
microtiter plate having a bottom composed of Gene
Screen Plus for 30 minutes at 25C. The microtiter
plate wells can then be washed three times for 5
minutes at 25C by adding 200 ~l of wash buffer
containing lX SSC, pH 7.0, and 0.17% Triton X-100, and
aspirating between washes.
C. p~tection
Detection can be accomplished by adding 200 ~l of
10 mM Tris, pH 7.00 to each sample and detecting the
amplified nucleic acid product detected by reflectance
fluorescence visualization.
