Note: Descriptions are shown in the official language in which they were submitted.
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; N~-0234
PROCESS FOR NUCLEIC ACID DE~ECTION
BY BINARY AMPLIFIC~TION
FI~LD OF I~VEtlTIO~l
This in~.~ention relates to the detection of
nucleic acid sequences and more specifically to a
process of combining the products f rom the
amplification of two portions of a target nucleic acid
sequence.
BACKGRO~ OF THE INV~TIOI~
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 ~or the
purpose of increasing the sensitivity nonradiometric
procedures In one general approach, improvements in
the total assay procedure have been examined, with
concomitant effects on the issues of complexity 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 Falkow,
describes a method of culturing cells to increase
their number and thus the amount of nucleic acid of
the organism suspected to be present, depositinq the
sample onto fixed support, and then contacting the
sample with a labeled probe, followed by washing the
support and detecting the label. One drawback to this
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
Z~3129~3
always successful. Maniatis et al., Mole~ular
Clonina: A L2boratorY 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 to 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 ou~ 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 of
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
amplification process can produce nucleic acid
sequences which are not faithful copies of the
original nucleic acid which was to be copied. These
3 . 20129~3
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., ~811, Vol. 12, 23-36 (1977),
describe a different hybridization format which
employs a two-step sandwich assay method employing
polynucleotide 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 is washed and a
second or reporter ~labeled) probe, also complementary
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
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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
EPA 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., employs a
capture probe and a reporter probe which hybridize to
distinct portions of the target nucleic acid. The
i5 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 reactlon which decreases
with increasing target nucleic acid length. Also,
sandwich nucleic acid probe assays, whether
heterogeneous two-step or one-step, or utilizing
solution hybridization, are not as sensitive as the
direct assay method.
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A disadvantage of all of these techniques relates
to the need to employ a specific hybridization step in
order to obtain the necessary specificity and thus
proper identification of the target nucleic acid
sequence of interest.
DI~CLOSURE OF THE INVEN~IQN
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 two specific nucleic acid
sequences contained within the preselected
nucleic acid sequence, said specific
nucleic acid sequences being positioned
such that when either sequence is amplified
under amplification conditions, the
extension product of either sequence cannot
serve as a template for the synthesis of
the other sequence,by
~1) treating the strands with two sets of
two oligonucleotide primers, one set
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 sets of primers
are selected so as to be sufficiently
complementary to the different strands
of each specific sequence to hybridize
therewith such that the extension
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products synthesized from one primer
from each of the two sets of primers,
when separated from their respective
complements, can serve as templates
for the synthesis of the extension
products of the other primer from each
of the two sets of primers; wherein
one of the primers of each set of
primers contains a LoxP sequence at
its S'-end;
(2) separating the primer extension
products from the templates on which
they were synthesized to produce
single-stranded molecules;
(3) treating the single-stranded molecules
generated from step ~2) with the two
sets of primers of step (1) under
conditions that primer extension
products are synthesized using each of
the single strands produced in step
(2) as templates; and
(4) repeating steps (2) and (3) to produce
sufficient primer extension products
for detection and/or measurement;
(C) treating the products of the two separate
amplification reactions by the recombinase
enzyme Cre; and
(D) detecting and/or measuring that product of
step (C) resulting from the two separate
amplification reactions.
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12~J:LT:D 12E;SCRI~ l OF THE INVENTIO
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, two
specific nucleic acid sequences, within the
preselected sequence, are amplified by first annealing
the denatured target nucleic acid present in the
sample with two sets of oligonucleotide primers
complementary to the specific nucleic acid sequences.
These primers are designed such that one primer of
each set of primers contains a LoxP sequence attached
to its 5'-end. The LoxP sequence is defined by a 34 -
nucleotide sequence
5'...ATAACTTCGTATAGCATACATTATACGAAGTTAT...3' . [See
Sternberg et al.,J. Mol. Biol., Volume 197, 197-212
~1986).] The specific nucleic acid sequences are
positioned such that when either sequence is amplified
under amplification conditions, the extension product
of either sequence cannot serve as a template for the
synthesis of the other sequence. During
amplification, each extension product formed from each
set of primers is complementary to one of the two
specific nucleic acid sequences within the preselected
nucleic acid sequence and is a template for further
primer extension. This process is then repeated as
necessary in order to produce the desired amount of
primer extenslon products for detection and/or
measurement.
b) Adding the recombinase enzyme Cre to the
products of the two separate amplification reactions,
allowing the enzyme to combine the amplification
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product from the first amplification with the
amplification product from the second amplification.
The resulting product is of a length of the
combination of the two specific nucleic acid sequences
plus 3q additional nucleotides.
c) Detecting the product of the above step by,
for examplel gel electrophoresis. Such detection can
differentiate between the desired combination product
on the one hand and the combination products formed
from the coupling of each amplification product with
itself.
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 minutes.
The Example below exemplifies the invention.
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EY~MPLE
Detection of HIV I
A. Amplification of Target Nucleic
Acid by PCR
The procedure as described in U.S. Patent
4,683,202 and in a product bulletin for GeneAmp DNA
Amplification Reagent Kit ~#N801-0043) can be foIlowed
utilizing the following specific conditions and
reagents. Two sequences of the HIV I genome can be
selected to be amplified. The first is a 103-
nucleotide base sequence located within the GAG pl7
region of HIV I, incorporated into a plasmid (the
plasmid incorporating most of the HIV I genome is
designated pBH10-R3), and can be amplified using
primers A and B as shown below:
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGGCAAGCAGGGAGCT
AGG..3'
Primer A
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTCTGAAGGGATGGTTGTA
CG..3'
Primer B
The second is a 160-base region also located within
the GAG pl7 region of HIV I, incorporated into a
plasmid (the plasmic incorporating most of the HIV I
genome is designated pBH10-R3), and can be amplified
using Primers C and D as shown below:
5'.... ....ATAACTTCGTATAGCATACATTATACGAAGTTATTTCCCTCAGACCCTTTTAG
TC..3'
erimer C
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGCGTACTCACCAGTCGC
CT..3'
Primer D
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Aliquots of serial dilutions ~lx10+7,
1X10+6,1X10~S~ 1X1014, lx10~3, lx10+2, lxlO+l, and zero
copies) of plasmid p~H10-R3 can be amplified using
PCR. Each aliquot can be combined with a buffer 200
~M in each of dA~P, dTTP, dCTP, and dGTP, 1.0 ~M in
each of Pri~ers A, B, C, and D, and containing 1 ~g of
human placental DNA/reaction and 2.5 units of a DNA
polymerase, in a total reaction volume of 100 ~1.
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.
The products of the amplification reactions can
be placed onto a 6% acrylamide gel run under standard
conditions. After electrophoresis, the gel can be
soaked in a 10 ~g/ml solution of ethidium bromide in
10 mM Tris, pH 7.0, for 15 minutes. The gel can then
be rinsed in 10 mM Tris, pH 7.0, and the resulting
product bands can be detected and/or measured by
irradiating the gel at 302 nm and visualizing the
fluorescent bands produced.
