Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-16
2001-04-03
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S051000, C422S051000, C422S067000, C422S001000, C435S006120, C435S007100, C435S287700, C435S287800, C435S287900, C436S518000, C436S541000, C436S807000, C436S810000
Reexamination Certificate
active
06210898
ABSTRACT:
BACKGROUND
This application relates to amplification of DNA and, in particular, to the simultaneous amplification of multiple target sequences using the Ligase Chain Reaction (hereinafter “LCR”). It is a continuation-in-part of co-owned and co-pending U.S. Ser. No. 07/860,702 filed Mar. 31, 1992, which is incorporated by reference.
This application is related to several other applications relating to LCR, including U.S. Ser. No. 131,936 filed Dec. 11, 1987, now pending, a continuation of that application, U.S. Ser. No. 720,739 filed Jun. 25, 1991, now pending, U.S. Ser. No. 470,674 filed Jan. 26, 1990, now abandoned; and a continuation-in-part from that application, U.S. Ser. No. 634,771 filed Jan. 9, 1991; now pending. It is noted that published EP-A-320 308 corresponds to U.S. Ser. No 131,936, and published EP-A-439 182 corresponds to U.S. Ser. No. 634,771. Both of the published documents in their entirety are incorporated herein by reference.
LCR is a method for amplifying exponentially the number of detectable target molecules. It involves the use of two pairs of probes. A first or primary pair hybridizes with one strand of a target sequence at near-adjacent positions so that they can be ligated together in template-dependent fashion to form a reorganized primary molecule. The secondary pair is capable of hybridizing to the reorganized primary molecule. LCR was first described by Backamn, et al. in EP-A-320 308. Much has been written about it since then. For example, see Wallace, EP-A-336 731; Orgel, WO 89/09835; Richards, WO 89/12696; Segev, WO 90/01069; and Barany,
Proc. Natl. Acad. Sci USA
88:189-193 (1991). A variation of LCR known as “Gap” LCR is described in EP-A439 182 and in Segev, WO 90/01069.
Instead of using two pairs of probes capable of forming blunt-ended duplexes, at least one probe of one of the probe pairs initially includes a “modified” end which renders the resultant duplex “nonblunt” and/or not a suitable substrate for the ligase catalyzed fusion of the two probe duplexes. A “modified end” has either (1) a blocking moiety (or additional base residues) on a group (e.g. the 5′ phosphate or the 3′ hydroxyl) which, under ordinary LCR conditions, obligatorily participates in the ligase catalyzed fusion or (2) omitted bases to create a “gap” between one probe terminus and the next probe terminus In the “gap” embodiment, modified ends are created by eliminating from one or more of the probes a short sequence of bases, thereby leaving a recess or gap between the 5′ end of one probe and the 3′ end of the other probe when they are both hybridized to the target (or target complement, or polynucleotide generated therefrom). In order for LCR to amplify the target, the gaps between the probes must be filled in (i.e., the modification must be “corrected”). In a first version, this can be done using a polymerase or a reverse transcriptase and an excess of deoxynucleotide triphosphates which are complementary to the target strand opposite the gap. Alternatively, this can be done by supplying a fifth probe complementary to the target and a sixth probe complementary to the fifth probe.
PCR or polymerase chain reaction is a different method for amplifying DNA. It employs two primers which hybridize to opposite strands of a double stranded target. A polymerase initiates extension of the primer using the target as a template by sequentially adding the appropriate complementary nucleotides. PCR is described in U.S. Pat. Nos. 4,883,195 and 4, 883,202, the entire disclosures of which are incorporated herein by reference.
PCR has been used in a multiplex manner to determine the presence of multiple target sequences in a single reaction. EP-A-364 255 describes the use of multiple primer sets to simultaneously amplify multiple target sequences by PCR. A similar disclosure is made in Chamberlain, et al.,
Nucl. Acids Res.,
16:1141-56 (1988).
In addition, Nickerson, et al.,
Proc. Natl. Acad. Sci. USA,
87:8923-8927 (1990) proposes an oligonucleotide ligation assay (“OLA”) for multiple target sequences, pending the development of “multiple, nonisotopic reporter groups”. OLA employs two contiguous probes that are ligated together and the ligated product is detected as a measure of the presence of a target sequence.
In spite of the existence of these disclosures, multiplex LCR is not available in the hands of the public. The an does not provide sufficient guidance actually to enable the concept of multiplex LCR. This is due largely to the inapplicability of PCR conditions to LCR.
SUMMARY OF THE INVENTION
Accordingly, we have now demonstrated the feasibility of multiplex LCR with as many as seven different probe sets. In one aspect, the invention is a method for performing LCR amplification simultaneously on two or more target sequences. The method comprises the steps of:
a. providing a reaction solution containing nucleic acid of a sample as single-stranded nucleic acid, said sample putatively having one or more of a plurality of target nucleic acid sequences;
b. for each putative target sequence, providing in the reaction solution at least four nucleic acid probes (a probe set), wherein: i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes; ii) the first probe is a single strand capable of hybridizing to a first segment of a primary stand of the target nucleic acid; iii) the second probe is a single strand capable of hybridizing to a second segment of said primary strand of the target nucleic acid; iv) the 5′ end of the first segment of said primary strand of the target is positioned relative to the 3′ end of the second segment of said primary strand of the target to enable joining of the first probe to the second probe when said probes are hybridized to said primary strand of said target nucleic acid, thus forming a reorganized primary molecule having a first portion and a second portion; v) the third probe is capable of hybridizing to a first portion of the reorganized primary molecule; and vi) the fourth probe is capable of hybridizing to a second portion of the reorganized primary molecule, the first portion of the reorganized primary molecule being positioned relative to the second portion of the reorganized primary molecule to enable joining of the third probe to the fourth probe when said third and fourth probes are hybridized to said reorganized primary molecule, thus forming a reorganized secondary molecule; and
wherein for each putative target sequence said probe set is provided at a concentration that enables said joining in the presence of each of the other probe sets, and
c. repeating the following cycle:
i) hybridizing said probes with nucleic acid in said sample;
ii) performing said joining to form said reorganized molecules; and
iii) denaturing nucleic acid in said sample;
whereby with successive cycles the quantity of reorganized primary and secondary molecules is increased for each putative target sequence present in the reaction solution.
In the usual case, the joining is performed by a ligase enzyme, or a ligase enzyme and a polymerase enzyme. Usually, the cycle of step c is repeated from 10 to 100 times, preferably from 20 to about 60 times.
Generally, the amplification product is detected by means of a unique detectable label associated with each probe set, each of said detectable labels being differentiable from the others. Labels preferably include specific binding members, such as haptens or polynucleotides. The labels can be used for either detection, or separation or both. In a preferred configuration, each of the probe sets are labeled with two distinct labels such that the reorganized molecules, when hybridized together, are labeled with two labels, at least one of which is a unique label and the other label is a common label, the same for each probe set.
In a preferred protocol, the common label is used for detection and the unique label is used to separate the reorganized molecules of one probe set from the reorganized molecules of at
Bouma Stanley R.
Gordon Julian
Hoijer Joanell
Jou Cynthia
Rhoads James
Abbott Laboratories
Jones W. Gary
Tung Joyce
Yasger Paul D.
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