WO1994002648A1

WO1994002648A1 - Gap-filling nucleic acid amplification and detection

Info

Publication number: WO1994002648A1
Application number: PCT/US1993/007066
Authority: WO
Original assignee: Imclone Systems Incorporated
Priority date: 1992-07-21
Filing date: 1993-07-21
Publication date: 1994-02-03
Also published as: AU4788493A

Abstract

This invention relates to a process for amplifying any desired specific nucleic acid sequence that exists in a nucleic acid or mixture thereof. The process comprises treating single strand RNA or separated complementary strands of DNA target with a molar excess of oligonucleotide complement pairs in which these oligonucleotide complement pairs have sequences complementary to the target, under hybridizing conditions wherein the gaps between the oligonucleotides bound to the same strand are filled in using no more than three nucleotides, followed by ligation of these oligonucleotides. The process uses oligonucleotide complement pairs selected so that the members of a pair have different melting temperatures. Oligonucleotide members with different melting temperatures permit the members to be selectively hybridized to the target sequence.

Description

"Gap-Filling Nucleic Acid Amplification and Detection" .

FIELD OF THE INVENTION

The present invention is directed to an improved process for amplifying and detecting existing nucleic acid sequences if they are present in a test sample.

BACKGROUND OF THE INVENTION

The present invention is an improvement of the repair chain reaction (RCR) process. RCR was discovered by David Segev and is described in PCT application US89/03125, which is

incorporated herein by reference. The proprietary interests in RCR are licensed to ImClone Systems Incorporated, New York, N.Y.

RCR involves detecting the presence of at least one single or double stranded target molecule in a sample. A single stranded target molecule comprises a nucleic acid target

sequence. A double stranded target molecule comprises a nucleic acid target sequence and a nucleic acid target complementary sequence. The RCR process of gap-filling plus ligation is analogous to the repair of mismatched bases, and other errors that occur during DNA replication, repair of UV damage, and other processes in vivo. The process comprises the steps of:

(a) treating the sample with a pair of

oligonucleotides for each strand of the target sequence and the target complementary sequence under hybridizing conditions,

(1) wherein each pair of oliyonucleotides is selected to hybridize either to the target sequence or the target complementary sequence; and

(2) wherein a gap of one or more bases is present between the members of each pair of oligonucleotides when the oligonucleotides are hybridized to the target sequence, or the target complementary sequence, with the proviso that filling the gap requires less than all four different types of bases;

(b) filling the gaps formed in step (a) with less than all four different types of bases complementary to the base or bases in the gap and joining the bases filling the gap to each other and to both adjacent hybridized oligonucleotides, thereby forming joined oligonucleotide products,

(1) wherein the joined oligonucleotide product hybridized to the target sequence comprises the target

complementary sequence; and

(2) wherein the joined oligonucleotide product hybridized to the target complementary sequence comprises the target sequence; (c) treating the hybridized joined oligonucleotide products of step (b) under denaturing conditions to separate the joined oligonucleotide products from the target sequence and the target complementary sequence to produce single-stranded

molecules;

(d) treating the single-stranded molecules produced in step (c) with a pair of oligonucleotides under hybridizing conditions, (1) wherein each pair of oligonucleotides comprises two oligonucleotides selected so as to be sufficiently complementary to the target sequence or to the target

complementary sequence to hybridize therewith; and (2) wherein a gap of one or more bases is present between the members of each pair of oligonucleotides when the oligonucleotides are hybridized to the target sequence or the target complementary sequence, with the proviso that filling the gap requires less than all four different types of bases ;

(e) filling the gaps formed in step (d) with less than all four different types of bases complementary to the base or bases in the gap and joining the base or bases filling the gap to each other and to both adjacent hybridized

oligonucleotides, thereby forming additional joined

oligonucleotide products, resulting in the amplification of the target sequence;

(f) treating the hybridized oligonucleotides of step (e) under denaturing conditions to separate the hybridized oligonucleotides and produce single-stranded molecules, wherein steps (d), (e) and (f) are repeated a desired number of times; and

(g) detecting the joined oligonucleotide product.

In a nucleic acid amplification and detection method, such as RCR, in which the members of an oligonucleotide pair are the same size or nearly the same size, both members of the pair generally have the same or very similar melting temperatures (Tm's). The melting temperature of an oligonucleotide, with respect to its complementary sequence, is primarily a function of the number of hydrogen bonds formed between the

oligonucleotide and its complementary sequence. The number of hydrogen bonds that an oligonucleotide can form is largely a function of its length. Oligonucleotide pairs of similar size are supplied in great excess in the reaction mixture of a process such as RCR. A problem with nucleic acid amplification reactions in which the oligonucleotides are of similar size is target independent hybridization of complementary

oligonucleotide probes and the formation of false product by blunt end ligation, especially at the beginning of the reaction. Formation of false product by blunt end ligation results in the loss of reliability and reproducibility.

The problem to be solved by the present invention is the improvement of the reliability and reproducibility of RCR. In prior nucleic acid amplification and detection methods, such as RCR, the advantage of using oligonucleotide pairs with different Tm's to selectively control the hybridization of the oligonucleotides was not recognized.

SUMMARY OF THE INVENTION

The above mentioned reliability and reproducibility problem of the prior art has been solved in the present invention, which relates to an improvement in the RCR process, as the RCR process is described in the Background Section above.

The improvement of the present invention comprises, prior to step (g), :

(h) selecting the pairs of oligonucleotides for step (a) and step (d) wherein,

(1) one member of each pair of oligonucleotides has a melting temperature, with respect to the target sequence or the target complementary sequence, that is sufficiently higher than the melting temperature, with respect to the target sequence or target complementary sequence, of the other member of the pair of oligonucleotides to cause selective hybridization of both oligonucleotides in each pair to the target sequence or target complementary sequence,

(2) wherein the member of each oligonucleotide pair with the higher melting temperature is denoted the higher Tm oligonucleotide and the member of each oligonucleotide pair with the lower melting temperature is denoted the lower Tm oligonucleotide; and

(i) selectively hybridizing the oligonucleotide pairs from step (h) to the target sequence or target complementary sequence by conducting steps (d), (e) and (f) under conditions alternating between high stringency, middle stringency and low stringency wherein, (1) under conditions of high stringency,

(A) the target molecule is denatured from the target complementary molecule; and

(B) all oligonucleotides are denatured from each other and from the target molecule; and

(2) under conditions of middle stringency,

(A) the higher Tm oligonucleotide member in each pair of oligonucleotides hybridizes to the target sequence or the target complementary sequence; and (B) the lower Tm oligonucleotide member in each pair of oligonucleotides does not hybridize to the target sequence or the target complementary sequence; and

(3) under conditions of low stringency the higher Tm oligonucleotide member and the lower Tm

oligonucleotide member in each pair of oligonucleotides both hybridize to the target sequence or the target complementary sequence; and (j) treating the hybridized oligonucleotides of step

(e) under conditions of high stridency to separate the

hybridized oligonucleotides and produce single-stranded

molecules, wherein steps (d)-(f) and (h)-(j) are repeated a desired number of times.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the steps of the process of the present invention as disclosed in Example 1.

Figure 2 is a photograph of an electrophoretic gel showing the experimental results of Method I in Example 1.

Figure 3 is a bar chart showing the experimental results of Method II in Example 1.

Figure 4 illustrates the steps of the capture assay

described in Method B in the Section entitled "Detection" of Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to an improved process for amplifying and detecting a target nucleic acid molecule in a test sample.

The process of the present invention can produce geometric amplification of a target nucleic acid molecule, provided that at least part of the nucleotide sequence is known in sufficient detail that complementary oligonucleotide pairs can be

synthesized. The target molecule can be in purified or non-purified form, and can be single stranded or double stranded DNA, single stranded or double stranded RNA or a DNA-RNA hybrid.

The target nucleic acid molecule contains the specific nucleotide sequence that hybridizes to members of the

oligonucleotide pairs. This sequence is called the target sequence. If a target nucleic acid molecule is double stranded, it will contain a target sequence and its complement called the target complementary sequence. The target sequence comprises at least as eight nucleotides, preferably at least twelve

nucleotides and more preferably at least sixteen nucleotides. There is no maximum number of nucleotides in the target sequence or target complementary sequence, which can constitute either a portion of the target molecule or the entire target molecule.

Any source of nucleic acid can be utilized as a source of the taxget nucleic acid molecule. For example, DNA or RNA isolated from bacteria, viruses, algae, protozoans, yeast, fungi, plasmids, cells in tissue culture and higher organisms such as plants or animals can be amplified with the process of the present invention. DNA or RNA from these sources may, for example, be found in samples of a bodily fluid from an animal, including a human, such as, but not limited to, blood, urine, lymphatic fluid, synovial fluid, bile, phlegm, saliva, aqueous humor, lacrimal fluid, menstrual fluid and semen. In addition, samples

containing DNA or RNA may, for example, be found in fluids from a plant, such as, but not limited to, xylem fluid, phloem fluid and plant exudates. Samples containing DNA or RNA may, for example, also be found in non-living sources such as, but not limited to, food, sewage, forensic samples, wet lands, lakes, reservoirs, rivers and oceans.

The improved process of the present invention is based on the fact that a longer oligonucleotide generally has a higher melting temperature (Tm) than a shorter oligonucleotide with respect to a target sequence or a target complementary sequence.

The use of oligonucleotide pairs whose members have

different melting temperatures permits the order in which the oligonucleotide members hybridize to the target sequence or the target complementary sequence to be selectively controlled. In the instant process, the greater the difference in the Tm of the members of the oligonucleotide pairs, up to a practical limit, the more the difference in the melting temperatures between the oligonucleotide pairs can be used to selectively hybridize the oligonucleotides to the target sequence or target complementary sequence. Controlling the hybridization of the different sized oligonucleotide members increases the reliability and

reproduciblity of the RCR amplification and detection process.

The term "melting temperature" or "Tm" as used herein refers to the temperature at which an oligonucleotide hybridizes to a complementary nucleic acid sequence to form a stable complex. The Tm of a given oligonucleotide is a function of various properties of the oligonucleotide, such as the size and composition of the oligonucleotide, as well as of the reaction medium, such as the concentration of the oligonucleotide, and the composition and pH of the reaction solvent. Usually, the oligonucleotide pairs of the present invention comprise one member that is longer than the other member. The longer member of each oligonucleotide pair generally has a higher Tm, with respect to its complementary sequence, than the shorter member of the oligonucleotide pair, with respect to its complementary sequence. Although, the longer member of the oligonucleotide pair is usually "the higher Tm oligonucleotide" and the shorter member of the oligonucleotide pair is usually "the lower Tm oligonucleotide," the relationship between length and Tm is not a necessary one. However, the general

relationship between the length of a nucleotide sequence and the melting temperature of the sequence permits the different oligonucleotides to be discussed in terms of their lengths as well as their melting temperatures. In the unusual event that the higher Tm oligonucleotide is shorter than the lower Tm oligonucleotide, the shorter Tm oligonucleotide is the higher Tm oligonucleotide.

An example of an oligonucleotide pair wherein the members have different Tm's is illustrated as follows:

5'TGCCGTAGACTGCCTATA -3' 5'-TACTCGAC-3' higher Tm oligonucleotide lower Tm oligonucleotide (See SEQ. ID. NOS. 1-2, respectively)

The members of an oligonucleotide pair are selected so that, under the conditions used in the amplification reaction, the Tm's are sufficiently different to cause selective

hybridization of each member of the pair to a target sequence or target complementary sequence. The terms "selectively

hybridize" or "selective hybridization" as used herein refers to the ability to control the order in which oligonucleotide members with different Tm's hybridize to the target sequence or the target complementary sequence in order to effect a

discernable improvement in the reliability and reproducibility of the RCR process.

Selective hybridization is primarily a function of the different melting temperatures of each member of an oligonucleotide pair with respect to the portion of the target sequence or the target complementary sequence to which it hybridizes. Shorter oligonucleotides generally require a smaller difference in length to effect selective hybridization than longer oligonucleotides. For example, when the lower Tm oligonucleotide has nine to twelve nucleotides, the higher Tm nucleotide should be at least two, preferably at least three and more preferably at least four nucleotides longer than the lower Tm oligonucleotide. When the lower Tm oligonucleotide has twelve to eighteen nucleotides, the higher Tm nucleotide should be at least three, preferably at least four and more preferably at least five nucleotides longer than the lower Tm

oligonucleotide.

The term "sufficiently different" as used herein refers to the difference in Tm between the members of an oligonucleotide pair that gives satisfactory selectivity in the improved RCR process of the invention.

Generally, melting temperatures of the members of an oligonucleotide pair are sufficiently different to cause

selective hybridization of the members to the target sequence or target complementary sequence if the difference in melting temperatures is at least 5°C, preferably at least 10°C, more preferably at least 15°C and most preferably at least 20°C.

The higher and lower Tm oligonucleotides that both

hybridize to either the target sequence or the target

complementary sequence each have complementary oligonucleotides. Accordingly, the higher Tm oligonucleotide and its complementary oligonucleotide constitute an oligonucleotide complement pair. Similarly, the lower Tm oligonucleotide and its complementary oligonucleotide constitute an oligonucleotide complement pair. The following generalized illustration depicts a higher Tm oligonucleotide with its complementary oligonucleotide and a lower Tm oligonucleotide with its complementary oligonucleotide. higher Tm oligonucleotide lower Tm oligonucleotide complement pair complement pair

5'-TAGCCGTAT-3' 5'-TGCCAA-3'

3'-ATCGGCATA-5' 3'-ACGGTT-5'

The oligonucleotides of the present invention may be characterized in two ways. One characterization of the

oligonucleotides is the grouping of the higher Tm and lower Tm oligonucleotides as an oligonucleotide pair, the members of which either both hybridize to a target sequence or both hybridize to a target complementary sequence. The other characterization of the oligonucleotides is the grouping of the higher Tm oligonucleotide with its complementary oligonucleotide and the lower Tm oligonucleotide with its complementary

oligonucleotide as oligonucleotide complement pairs. As in the classical RCR process, the oligonucleotide pairs of the present invention are designed so that a gap of at least one nucleotide base is formed between the members of the

oligonucleotide pair when both members are hybridized to the target sequence or target complementary sequence.

In the improved process of the present invention, the space that is created between the higher Tm and the lower Tm members of an oligonucleotide pair when they hybridize to a target sequence or to a target complementary sequence is referred to as a gap. The oligonucleotide pairs are selected so that there is a gap in the nucleotide sequence of at least one base,

preferably two or more bases such as at least three, four, six or ten bases, between the two oligonucleotides when the two oligonucleotides are hybridized with the target sequence. The maximum size of the gap is limited only by the length of the segment of the target sequence that has less than all four types of nucleotide bases. In the present invention, the gap may be filled before the lower Tm oligonucleotide hybridizes to the target strand, so that the gap may not be actually formed.

Nevertheless, the space on each target sequence, target

complementary sequence or joined oligonucleotide product between the hybridizing oligonucleotides will be referred to as the gap. More than two oligonucleotide pairs, resulting in more than two gaps, may be used in the process of the present invention. The gap is filled with less than all four types of

nucleotide bases. The term "less than all four nucleotide bases" as used herein refers to any combination of the

complementary deoxyribonucleotide bases adenosine, thymidine, guanine or cytosine wherein fewer than all four types of bases, i.e. one, two or three, but not all four types of bases, are used in the method to fill the gap. The ribonucleotide uracil can be substituted for thymidine in the method of the present invention. The term "gap-filling" as used herein refers to the

classical RCR process of filling in the gap. For each

oligonucleotide pair, one member acts as a primer in the

polymerization reaction that fills the gap and the other member becomes ligated to the polymerization extension product. The member that acts as a primer is designated the priming

oligonucleotide member. Similarly, the member that becomes ligated to the polymerization extension product is designated the ligation oligonucleotide member. The ligation

oligonucleotide member is phosphorylated at its 5' end.

In a preferred embodiment of the process of the present invention, once the priming oligonucleotide member hybridizes to the target sequence or target complementary sequence, the gap-filling process begins with the addition by polymerization of a nucleotide that is complementary to the first base in the gap of a target sequence, or target complementary sequence, to the priming oligonucleotide member. If the gap is larger than one nucleotide base, additional nucleotide bases are added by polymerization. The addition of nucleotides ends when a base on the target sequence, or target complementary sequence, is reached that does not have a complementary base supplied in the reaction mixture. Either before, during or after the gap-filling process, the ligation member of the oligonucleotide pair hybridizes to the target sequence or target complementary sequence, if present, immediately adjacent to the gap. The last nucleotide used to fill the gap and the ligation oligonucleotide member are then ligated to form a single stranded nucleic acid termed a "joined oligonucleotide product."

The term "joined oligonucleotide product" as used herein refers to the single stranded nucleic acid sequence that is formed when the nucleotides that fill the gap are joined to the oligonucleotides that define the gap by the process of the present invention. The joined oligonucleotide product that hybridizes to the target sequence comprises the sequence of the target complementary sequence. Similarly, the joined

oligonucleotide product that hybridizes to the target

complementary sequence comprises the sequence of the target sequence. Accordingly, reference to the target sequence or the target complementary sequence in the specification and the claims of the present invention also includes joined

oligonucleotide products that have hybridized to and been formed from the target or target complementary sequences.

Generally, the gap-filling and ligation steps occur in a buffered aqueous solution, preferably at a pH of 7-9, most preferably at pH 7.5. The oligonucleotide complement pairs will be present in molar excess of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶, pairs per nucleic acid target sequence. The exact amount of the pairs to be used in diagnostic purposes may not be known due to uncertainty as to the amount of the nucleic acid target in a sample. However, using an average amount of 10¹⁵ oligonucleotide complement pairs is applicable in a typical diagnosis assay format. A large molar excess is preferred in any case to improve the efficiency of the process of the invention.

Under conditions of low stringency, the lower Tm members of the oligonucleotide complement pairs hybridize not only to the target nucleic acid molecule or joined oligonucleotide product, but to any unhybridized higher Tm members of the oligonucleotide complement pairs as well. Hybridization of the lower Tm members of the oligonucleotide complement pairs to the higher Tm members of the pairs reduces the number of lower Tm oligonucleotides that are available to hybridize to nucleic acid molecules having the target sequence or target complementary sequence, as the case may be. In order to increase the efficiency of the process, the lower Tm oligonucleotide members are provided at a higher concentration than the higher Tm oligonucleotide members. The lower Tm oligonucleotides are preferably provided at a

concentration at least 1.1 times greater than the higher Tm oligonucleotides, more preferably at least 1.5 times greater than the higher Tm oligonucleotides and most preferably at least 1.75 times greater than the higher Tm oligonucleotides.

There is a limit to the number of times that the

concentration of the lower Tm members can be increased relative to the concentration of the higher Tm members for a given oligonucleotide complement pair. The maximum increase in concentration of the lower Tm members relative to the higher Tm members is dependent primarily on the differential between the melting temperatures of the lower Tm and higher Tm members of an oligonucleotide complement pair. The differential in melting temperature between members of an oligonucleotide complement pair is dependent on factors such as, but not limited to, the length of the members, the composition of the members and the composition of the reaction mixture. In general, the higher the differential in Tm's between the members of an oligonucleotide complement pair, the higher the limit to the number of times that the concentration of the lower Tm oligonucleotide members can be increased relative to the concentration of the higher Tm members. The maximum increase in concentration of the lower Tm oligonucleotides relative to the higher Tm oligonucleotides is preferably at most ten times greater than the concentration of the higher Tm oligonucleotides, more preferably at most six times greater than the concentration of the higher Tm

oligonucleotides, even more preferably at most four times greater than the concentration of the higher Tm oligonucleotides and most preferably at most two times greater than the

concentration of the higher Tm oligonucleotides. The term "overhang" as defined herein is used to describe that portion of an oligonucleotide complement pair wherein one member of the pair has several bases that protrude beyond the other member of the pair.

An overhang can be at the 3' or 5' end of the particular oligonucleotide member, or at both the 3' and the 5' ends. For example:

The bases in the overhang may protrude into the area of the gap, or into the area of an oligonucleotide on the side away from the gap. For example:

Overhangs on the side of the gap

Overhangs on the side away from the gap

The terms "overlapping gap" and "non-overlapping gap" as used herein refers to the embodiment in which two

oligonucleotide pairs are selected so that the position of the gap on one target strand relative to the gap on the

complementary target strand can result in an overlap of the gaps or a non-overlap of the gaps. For example:

Overlapping Gap with Blunt Ends

Overlapping Gap with Overhangs

Non-Overlapping Gap with Overhangs

In the improved process of the present invention, the number of bases in the overhang of the oligonucleotide pairs is sufficient to cause a sufficient difference in the Tm of each member of an oligonucleotide pair so that the hybridization of each member of the pair to its respective complementary sequence on the target sequence or the target complementary sequence is selectively controlled. Preferably the overhang has at least two bases, more preferably at least six bases and most

preferably nine or more bases. The different melting

temperatures of the two complementary nucleotide pairs, A-T and C-G, are important elements to consider in the design of the oligonucleotide pairs. The embodiment of the present invention wherein two oligonucleotide complement pairs each have 3 ' overhangs that form a non-overlapping gap will now be illustrated.

The oligonucleotide complement pairs are illustrated as follows:

In the amplification process, a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions. The process proceeds in the presence of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶ molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, G), a heat-stable

polymerase and heat-stable ligase.

The process is begun by subjecting a single or double stranded target molecule and the oligonucleotide complement pairs to conditions of high stringency, usually high

temperature, to denature all of the nucleic acids to each other.

As the temperature is reduced to effect conditions of middle stringency, the higher Tm members of each oligonucleotide complement pair, A and D, selectively hybridize to the target sequence and target complementary sequence, respectively. The gap filling process is then initiated by a polymerase at the 3' end of the hybridized oligonucleotides. As the temperature is further reduced to effect conditions of low stringency, the lower Tm members of each oligonucleotide complement pair, B and C, selectively hybridize to the target sequence and target complementary sequence, respectively, at the site where the gap, which will now usually be filled, ends. The sequence that fills each gap and the lower Tm oligonucleotides are then ligated to form joined oligonucleotide products in the presence of a ligase. The process is repeated to achieve the desired level of amplification. (See Figure 1 and Example 1). In another embodiment of the present invention two

oligonucleotide complement pairs with 5' overhangs form a non-overlapping gap.

The oligonucleotide complement pairs are illustrated as follows:

In the amplification process of this embodiment, a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under conditions of high

stringency, i.e. high temperature. The process proceeds in the presence of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶ molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of

nucleotide bases (A, C, G), a heat-stable polymerase and heat-stable ligase.

As the temperature is reduced to effect conditions of middle stringency, the higher Tm members of each oligonucleotide complement pair, F and G, selectively hybridize to the target sequence and target complementary sequence, respectively. As the temperature is further reduced to effect conditions of low stringency, i.e. low temperature, the lower Tm members of each oligonucleotide complement pair, E and H, selectively hybridize to the target sequence and target complementary sequence, respectively. The gap filling process is then initiated by a polymerase at the 3' end of the lower Tm oligonucleotides, E and H. The sequence that fills the gap and the higher Tm

oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.

In another embodiment of the present invention, one member of an oligonucleotide pair has a 3' overhang and the equivalent member of the other pair has a 5' overhang that form an

overlapping gap.

The oligonucleotide complement pairs are illustrated as follows:

In the amplification process of this embodiment, a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature. The process proceeds in the presence of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶, molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, T), a heat-stable polymerase and heat-stable ligase.

As the temperature is reduced to effect conditions of middle stringency, the higher Tm members of each oligonucleotide complement pair, J and K, selectively hybridize to the target sequence. The gap filling process is then initiated by the polymerase at the 3' end of the oligonucleotide J. As the temperature is further reduced to effect conditions of low stringency, i.e. low temperature, the lower Tm members of each oligonucleotide complement, L and M, selectively hybridize to the target complementary sequence. The gap filling process is then initiated by a polymerase at the 3' end of the

oligonucleotide M. The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.

In another embodiment of the present invention, one member of the pair has a 5' overhang and the equivalent member of the other pair has a 3' overhang that form an overlapping gap.

The oligonucleotide complement pairs are illustrated as follows :

In the amplification process of this embodiment, a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature. The process proceeds in the presence of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶ molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, T), a heat-stable polymerase and heat-stable ligase.

As the temperature is reduced to effect conditions of middle stringency, the higher Tm members of each oligonucleotide complement pair, P and Q, selectively hybridize to the target complementary sequence. The gap filling process is then

initiated by the polymerase at the 3' end of the oligonucleotide Q. As the temperature is further reduced to effect conditions of low stringency, i.e. low temperature, the lower Tm members of each oligonucleotide complement pair, N and O, selectively hybridize to the target sequence. The gap filling process is then initiated by the polymerase at the 3' end of the

oligonucleotide N. The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.

In another embodiment of the present invention, both members, R and T, of one oligonucleotide complement pair are higher Tm oligonucleotides and both members, S and U, of another oligonucleotide complement pair are lower Tm oligonucleotides.

The oligonucleotide complement pairs are illustrated as follows:

In the amplification process of this embodiment, a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature. The process proceeds in the presence of about 10⁵-10¹⁸, preferably 10⁹-10¹⁶ molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases, a heat-stable polymerase and heat-stable ligase. As the temperature is reduced to effect conditions of middle stringency, the higher Tm members of each oligonucleotide complement pair, R and T, selectively hybridize to the target sequence and the target complementary sequence, respectively. The gap filling process is then initiated by the polymerase at the 3' end of the oligonucleotide R. As the temperature is further reduced to effect conditions of low stringency, the lower Tm members of each oligonucleotide complement pair, S and U, selectively hybridize to the target sequence and the target complementary sequence, respectively. The gap filling process is then initiated by the polymerase at the 3' end of the

oligonucleotide U. The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.

The same conditions or routine modifications apply to each of these embodiments irrespective of whether no overhangs , 3 ' or 5' overhangs, or overlapping or non-overlapping gaps are used. If heat-stable gap-filling and sealing agents are used, such as thermostable polymerase and ligase, a heat-stable polymerase and ligase could be extracted from Thermus

thermophilus. Such a heat-stable polymerase is described by A.D. Kaledin, et al., Biokhimiya, 45, 644-51 (1980). A heatstable ligase is described by M. Takahashi et al., J. Biol.

Chem., 159 (16), 10041-10047 (1984).

The invention will now be illustrated by a specific

example. In conjunction with the general and detailed

description above, the example provides further understanding of the present invention and outlines some aspects of the preferred embodiments of the invention. The scope of the present

invention is not limited by the example. EXAMPLE 1

This example illustrates the gap filing-ligation embodiment to amplify and detect target DNA sequences of hepatitis B virus (HBV) using oligonucleotide complement pairs having 3' overhangs that form a non-overlapping gap. (See Figure 1)

Two sources of HBV DNA are used in this example. The first source is a 3.2 Kb nucleic acid sequence containing the HBV-ayw genome. The second source is a series of clinical serum samples suspected to contain HBV. The HBV target source is the region of the genome spanning nucleotides 2429-2480 having the

sequence: 5' AAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCCTTGGACTCATAAGGT 3' 3' TTCTAGAGTTAGAGCCCTTAGAGTTACAATCATAAGGAACCTGAGTATTCCA 5'

See SEQ. ID. NOS. 3-4 The gaps formed when the respective oligonucleotide

complement pairs are hybridized to each of the target sequences are underlined above.

The two oligonucleotide complement pairs used in this embodiment, A-C and B-D, each have one member of the pair that has a Tm that is different than the other member of the pair.

Oligonucleotides A and D have higher Tm's than oligonucleotides

C and B, respectively. The melting temperature (Tm) of each oligonucleotide is as follows:

Oligonucleotide Tm

1 ) A 58 .5°C

2 ) B 40 . 0°C

3 ) C 43.2°C

4 ) D 52 .5°C

In each of the complementary oligonucleotide pairs , there are 3' overhung bases. The 5' and 3' ends of oligonucleotides D and C, respectively, are protected with an amino linker arm. The 5' end of oligonucleotide A is labeled with Fluorescein. The 3' end of oligonucleotide B is labeled with Biotin. The 5' ends of oligonucleotides B and C are phosphorylated. The construction of the oligonucleotides is as follows:

See SEQ ID NO'S. 5-8 (A, B, D and C respectively).

When these oligonucleotide complement pairs are

hybridized to the HBV target sequence, a gap exists between each pair as illustrated:

I. Procedure

Materials::

Each 25 μl aliquot of reaction mixture contains the following:

50 ng of each of the four synthetic oligonucleotide members; A-Fluorescein; B-Biotin; C; D.

15 units of Ampligase^™ heat-stable ligase (Epicenter). 1.5 units of Ampli-Taq^™ heat-stable DNA polymerase

(Perkin-Elmer). in: 20 mM Tris-HCl pH 7.6, 50 mM KCl, 10.0 mM MgCl₂,

1.0 mM NAD, 10 mM DTT, 0.2 mM dGTP, 0.2 mM of dATP and 0.2 mM TTP, 0.1 mM EDTA.

Method I: Amplification of the HBV samples for detection on an electrophoretic gel.

Clinical serum samples suspected of containing HBV are treated with proteinase K prior to analysis with the method of the present invention as follows: 10 μl of proteinase K at 10 mg/ml is added to 90 μl of each serum sample for a final proteinase K concentration of 1mg/ml. The serum samples are incubated for three (3) hours at 37°C, followed by an incubation for ten (10) minutes at 95°C. The serum samples are spun down in a microfuge for ten (10) minutes. The supernatant from each tube is collected and used as the source of HBV target DNA for amplification.

25 μl of reaction mixture is added to each of 12

microfuge tubes (500 μl size).

To the first tube is added 1 μl of a control sample

(negative control) containing 0 ng of DNA in distilled water (dH₂O). To each successive tube is added 1 μl of a serial dilution of HBVp3.2 or a Proteinase K treated clinical serum sample suspected of containing HBV: Tube DNA Contents

1 Control, no target

2 HBVp3.2 1 pg

3 HBVp3.2 100 fg

4 HBVp3.2 10 fg

5 HBVp3.2 1 fg

6 Serum Sample 1

7 Serum Sample 2

8 Serum Sample 3

9 Serum Sample 4

10 Serum Sample 5

11 Serum Sample 6

12 Serum Sample 7

Method II: Titration of HBV samples for detection with a capture assay.

25 μl of reaction mixture is added to each of 5 microfuge tubes (500 μl size).

To each successive tube is added 1 μl of a sample of HBVp3.2 in the following amounts:

Tube DNA Contents

1 Control, no target

2 HBVp3.2 1000 fg

3 HBVp3.2 100 fg

4 HBVp3.2 10 fg

5 HBVp3.2 1 fg

Each of the tubes is covered with 25 μl of light mineral oil to prevent evaporation. The tubes are placed in a DNA Thermal Cycler (Perkin Elmer Model 4800).

The tubes are incubated in cycles as follows:

94°C for 2 seconds

62°C for 2 seconds

44°C for 2 seconds

The set of tubes from Method I are cycled for 32 cycles.

The set of tubes from Method II are cycled for 27 cycles. All tubes are then placed at 4°C until ready for

analysis.

After the cycling is completed, the contents of each of the tubes from Method I are analyzed on a gel and the contents of each of the tubes from Method II are analyzed in a capture assay.

Detection:

Method A. Sizing on a Gel.

The reaction products are separated by size using non-denaturing polyacrylamide gels. 8 μl aliquots of solution from each tube is loaded on a 12% polyacrylamide gel (6 cm. long) using Tris-borate buffer (pH 8.5). Electrophoresis is carried out at 20 volts/cm for three hours, after which the gel is stained with ethidium bromide (1 μg/ml). The gel is then photographed. The photograph is shown in Figure 2.

Method B. Non-radioactive capture assay format

(Fluorescein and Biotin). (See Figure 4)

Procedure:

1. The wells of a microtiter plate (Immulon II) are coated with 10 μg of egg white avidin (Precision Chemicals) in 100 μl of PBS and incubated overnight at 4°C. 2. The wells of the microtiter plate are washed two times with PBS/0.5% Tween (PBST). The wells are further coated by adding 100 μl of 2% BSA in PBS to each well for 30 min. at room temperature (RT). The blocking solution is decanted and the wells are washed 4 times with PBST. To further reduce background signals, the wells of the microtiter plate are incubated with 0.01% H₂O₂ for 30 minutes at RT.

3. Following incubation of the reaction tubes, 6 μl of each amplified sample (reaction mixture) is adjusted, with gentle mixing, to a volume of 100 μl with PBS. 50 μl of the adjusted reaction mixture is transferred from each reaction tube to a separate blocked well on the microtiter plate. The mixtures in the microtiter wells are incubated for one hour at room temperature.

4. The diluted reaction mixtures are decanted from the microtiter wells. The wells of the plate are washed five times with PBST. A horseradish peroxidase conjugated rabbit anti-fluorescein antibody (Biodesign) is diluted 1:10,000 in PBS. 100 μl of the conjugate is added to each well of the microtiter plate and incubated for one hour at room

temperature. 5. The conjugate solution is decanted from the

microtiter wells. The wells are washed five times with PBST. 100 μl of trimethyl benzidine (TMB) and hydrogen peroxide (1:1 vol.) substrate solution is added to each well of the

microtiter plate and incubated for thirty minutes at room temperature. 100 μl of 1N H₂SO₄ is added to each well to stop the reaction. The optical density (O.D.) of each well is read at 450 nm, using a microtiter plate reader. The results are shown in Figure 3.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Grimberg, Jacob

(ii) TITLE OF INVENTION: Improved Process for Amplifying and

Detecting Nucleic Acid Sequences

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ImClone Systems Incorporated

(B) STREET: 180 Varick Street

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10014

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/917,657

(B) FILING DATE: 21-JUL-1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Feit, Irving N.

(P) REGISTRATION NUMBER: 28,601

(C) REFERENCE/DOCKET NUMBER: GRI-1-PT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-645-1405

(B) TELEFAX: 212-645-2054

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TGCCGTAGAC TGCCTATA 18 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TACTCGAC 8

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AAGATCTCAA TCTCGGGAAT CTCAATGTTA GTATTCCTTG GACTCATAAG GT 52 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ACCTTATGAG TCCAAGGAAT ACTAACATTG AGATTCCCGA GATTGAGATC TT 52

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

AAGATCTCAA TCTCGGGAAT CTCAAT 26 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CCTTGGACTC ATAAGGT 17 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ACCTTATGAG TCCAAGGAAT ACTAAC 26 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

CCCGAGATTG AGATCTT

Claims

CLAIMS WHAT IS CLAIMED IS:

1. In an improved process for amplifying and detecting, in a sample, a single stranded target nucleic acid molecule comprising a target sequence or a double stranded target nucleic acid molecule comprising a target sequence and a target complementary sequence, the process comprising the steps of:

(a) treating the sample with a pair of

oligonucleotides for each strand of the target sequence and the target complementary sequence under hybridizing

conditions,

(1) wherein each pair of oligonucleotides is selected to hybridize either to the target sequence or the target complementary sequence; and

( 2 ) wherein a gap of one or more bases is present between the members of each pair of oligonucleotides when the oligonucleotides are hybridized to the target

sequence, or the target complementary sequence, with the proviso that filling the gap requires less than all four different types of bases;

(b) filling the gaps formed in step (a) with less than all four different types of bases complementary to the base or bases in the gap and joining the bases filling the gap to each other and to both adjacent hybridized

oligonucleotides, thereby forming joined oligonucleotide products,

complementary sequence; and (2) wherein the joined oligonucleotide product hybridized to the target complementary sequence comprises the target sequence;

(c) treating the hybridized joined oligonucleotide products of step (b) under denaturing conditions to separate the joined oligonucleotide products from the target sequence and the target complementary sequence to produce single-stranded molecules; (d) treating the single-stranded molecules produced in step (c) with a pair of oligonucleotides under hybridizing conditions,

( 1 ) wherein each pair of oligonucleotides comprises two oligonucleotides selected so as to be

sufficiently complementary to the target sequence or to the target complementary sequence to hybridize therewith; and

(2 ) wherein a gap of one or more bases is present between the members of each pair of oligonucleotides when the oligonucleotides are hybridized to the target

sequence or the target complementary sequence, with the proviso that filling the gap requires less than all four different types of bases;

oligonucleotides, thereby forming additional joined

(f) treating the hybridized oligonucleotides of step (e) under denaturing conditions to separate the hybridized oligonucleotides and produce single-stranded molecules, wherein steps (d), (e) and (f) are repeated a desired number of times; and (g) detecting the joined oligonucleotide product; the improvement comprising, prior to step (g),: (h) selecting the pairs of oligonucleotides for step (a) and step (d) wherein,

(1) one member of each pair of oligonucleotides has a melting temperature, with respect to the target sequence or the target complementary sequence, that is sufficiently higher than the melting temperature, with respect to the target sequence or target complementary

sequence, of the other member of the pair of oligonucleotides to cause selective hybridization of both oligonucleotides in each pair to the target sequence or target complementary sequence,

(i) selectively hybridizing the oligonucleotide pairs from step (h) to the target sequence or target

complementary sequence by conducting steps (d), (e) and (f) under conditions alternating between high stringency, middle stringency and low stringency wherein, (1) under conditions of high stringency,

(A) the target molecule is denatured from the target complementary molecule; and (B) all oligonucleotides are denatured from each other and from the target molecule; and

(2) under conditions of middle stringency, (A) the higher Tm oligonucleotide member in each pair of oligonucleotides hybridizes to the target sequence or the target complementary sequence; and (B) the lower Tm oligonucleotide member in each pair of oligonucleotides does not hybridize to the target sequence or the target complementary sequence; and

oligonucleotide member in each pair of oligonucleotides both hybridize to the target sequence or the target complementary sequence; and (j) treating the hybridized oligonucleotides of step (e) under conditions of high stringency to separate the hybridized oligonucleotides and produce single-stranded molecules, wherein steps (d)-(f) and (h)-(j) are repeated a desired number of times.

2. The process of claim 1, wherein the gaps on the complementary target sequence and the joined oligonucleotide product overlap.

3. The process of claim 1, wherein the gaps on the complementary target sequence and the joined oligonucleotide product do not overlap.

4. The process of claim 1, wherein the gap contains at least two nucleotides.

5. The process of claim 1, wherein each oligonucleotide pair has one member that overhangs the other member of the pair wherein the number of bases in the overhang of the oligonucleotide pair is sufficient to cause a sufficient difference in the melting temperature of each member of the oligonucleotide pair so that the hybridization of each member of the pair to its respective complementary sequence on the target sequence or target complementary sequence is selectively controlled.

6. The process of claim 1, wherein at least one oligonucleotide complement pair has a 3' overhang.

7. The process of claim 1, wherein at least one oligonucleotide complement pair has a 5' overhang.

8. The process of claim 1, wherein at least one oligonucleotide complement pair has both 5' and 3' overhangs.

9. The process of claim 1, wherein the higher Tm oligonucleotide member functions as a priming oligonucleotide for gap-filling in step (e).

10. The process of claim 1, wherein the lower Tm

oligonucleotide member functions as a ligation oligonucleotide for gap-filling in step (e).

11. The process of claim 1, wherein the lower Tm

oligonucleotide member is provided at a concentration 1.1 times greater than the higher Tm oligonucleotide member.

12. The process of claim 1, wherein the lower Tm oligonucleotide member is provided at a concentration 1.5 times greater than the higher Tm oligonucleotide member.

13. The process of claim 1, wherein the lower Tm oligonucleotide member is provided at a concentration 1.75 times greater than the higher Tm oligonucleotide member.