HK1013926A - Method for amplifying specific nucleic acid sequences - Google Patents

Description

Method for amplifying specific nucleic acid sequence

Technical Field

The present invention relates to a method for amplifying specific nucleic acid target sequences in vitro. In particular, the invention relates to methods for the thermotolerant restriction endonuclease-mediated selective amplification of nucleic acid target sequences containing sequence differences, including point mutations, deletions and insertions.

Background

A variety of genetic and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Some of these variations are directly related to the presence of disease, while others are related to disease risk and/or prognosis. There are 500 human genetic diseases due to single gene mutations, these include cystic fibrosis, muscular dystrophy, alpha 1-antitrypsin deficiency, phenylketonuria, sickle cell anemia or traits, and various other hemoglobinopathies. In addition, individuals with increased susceptibility to several common polygenic states, such as atherosclerotic heart disease, have been shown to be genetically related to specific DNA sequence polymorphisms. Cancer is thought to be caused by accumulation of lesions involving cell proliferation or differentiation genes. The ras proto-oncogenes K-ras, N-ras and H-ras and the p53 tumor suppressor gene are examples of genes that are frequently mutated in human cancers, and specific mutations in these genes result in activated or increased transforming capacity. Genetic analysis is likely to become a clinically routine analysis for assessing disease risk, disease diagnosis, predicting patient prognosis or response to treatment, and for monitoring disease progression. The introduction of such genetic tests relies on the development of simple, inexpensive and rapid methods for genetic variation analysis.

In rare cases, a mutation can be detected if it happens to be located within the natural restriction endonuclease recognition/cleavage site. WO84/01389 describes a method for distinguishing between wild-type genes and non-wild-type mutants by screening for the presence or absence of restriction endonuclease sites. The inventors have illustrated this principle by analyzing the variant sequence at codon 12 of the human H-ras proto-oncogene. Codon 12 of the wild-type sequence forms part of the recognition/cleavage sites for the restriction endonucleases NaeI and HpaII. Digestion with these endonucleases can distinguish between a wild-type proto-oncogene and an activated oncogene having a mutation at this codon. Point mutations at H-ras codon 12 are frequently found in bladder cancer and this strategy may form the basis of a medical diagnostic screening kit.

Nucleic acid in vitro amplification methods have been widely used in genetics and disease diagnosis. Polymerase Chain Reaction (PCR) is a powerful sensitive method for the in vitro amplification of specific nucleic acid fragments (R.K. Saiki et al, 1985, science 230, 1350-. PCR is mediated by oligonucleotide primers that flank the target sequence to be synthesized and are complementary to sequences located on opposite strands of the template DNA. These steps occur in the reaction due to temperature cycling (thermal cycling). The template DNA is first denatured by heating, and the reaction is then cooled to allow the primers to anneal to the target sequence, which are then extended by a DNA polymerase. The cycle of denaturation, annealing and DNA synthesis is repeated multiple times and the product of each round of amplification is used as a template for the next round. This process results in exponential amplification of amplicons that incorporate oligonucleotide primers at the 5' termini and contain newly synthesized copies of the sequences located between the primers.

PCR is extremely versatile and many modifications of this basic method have been developed. Primers used in PCR may be perfectly matched to the target sequence or they may contain mismatched or modified bases. Additional sequences at the 5' end of the primers may facilitate the acquisition of PCR amplicons and the inclusion of labeled primers may facilitate detection. The inclusion of mismatched bases in the primer may result in the creation of new restriction endonuclease recognition/cleavage sites, which may be located entirely within the primer sequence, or they may span sequences located partially within the primer and partially within the newly synthesized target sequence (J.B. and A.D. Levinson (1988), Nature 334, 119-. General rules for designing primers containing mismatched bases near the 3' end have been established (S.Kwok et al (1990), nucleic acids research 18, 999-.

Modified primers containing mismatched bases were used to introduce a new recognition/cleavage site for restriction endonucleases into the H-ras amplicon mutated at codon 12 (R.Kumar and M.Barbacid (1988), oncogene 3, 647-651). Similarly, primers containing mismatched bases are used in a process called allele-specific enrichment (Todd AV et al leukemia, 1991, 5: 160) or enrichment PCR (Levi S et al, cancer research, 1991, 6: 1079), which are very sensitive methods for detecting point mutations. In these methods, if the sequence is wild-type at codon 12, the DNA sample is amplified with primers that introduce an EcoNI site in the N-ras amplicon, or a BstNI site in the K-ras amplicon. Aliquots of the PCR reactions were digested with the appropriate restriction endonucleases to cleave the wild-type amplicon before re-amplifying the digestion-resistant amplicon in a second round of PCR cycles. These methods result in preferential amplification of sequences with point mutations at codon 12 in ras. Recently, a simplified enrichment PCR method was disclosed (Singh et al, J. International cancer sciences, 1994; 5: 1009) that allows the reaction to be performed in a single tube, which also requires an initial round of PCR amplification, however, restriction endonucleases are then added directly to the reaction tube. After incubation with restriction nucleases, a second round of PCR cycles results in amplification of sequences with mutations within the restriction endonuclease recognition/cleavage sites. This analysis of native or induced restriction endonuclease sites in the PCR amplicon requires subsequent DNA polymerase activity for PCR followed by restriction endonuclease activity for cleavage analysis. Enrichment PCR methods first require DNA polymerase activity for PCR, followed by restriction endonuclease activity to cleave specific sequences, followed by further DNA polymerase activity to re-amplify the digestion resistant amplicons.

The ability to simultaneously utilize the activities of a restriction endonuclease and a DNA polymerase in a PCR process may provide several advantages that may allow for the creation of a simple method for the separate or preferential amplification of variant sequences in reactions containing all reagents, including enzymes, at the start of PCR. It was not previously known whether the inclusion of a restriction endonuclease in a PCR would result in (i) complete (or partial) inhibition of amplification of a sequence containing a restriction endonuclease recognition/cleavage site and (ii) amplification of a variant of a sequence lacking a restriction endonuclease recognition/cleavage site alone (or preferentially). The ability to completely inhibit sequence amplification and/or to amplify variant sequences alone may result in a method that does not require further manipulation prior to analysis. The reduced number of steps required for selective amplification and/or subsequent analysis of amplicons may result in faster, less labor intensive, and/or more amenable to automated methods. A further advantage is that the reaction will be done in a closed system and this will reduce the chance of contamination during the PCR process.

Such a method would require both the activity of a restriction endonuclease and a DNA polymerase under conditions compatible with PCR. Such restriction endonucleases and DNA polymerases must i) function in the same reaction conditions (e.g., salt, PH) that must be compatible with PCR and ii) be sufficiently thermally stable in these reaction conditions to maintain activity during the cycles required for PCR. Restriction endonucleases suitable for use in conjunction with PCR must be active at temperatures compatible with the stringent conditions under which the primers anneal during PCR, typically 50 ℃ to 65 ℃. The simultaneous activity of thermostable DNA polymerases and restriction endonucleases has previously been developed for mediating in vitro amplification in an isothermal reaction called strand displacement amplification (EPO384315 AI). It was not previously known whether a restriction endonuclease would be sufficiently thermostable to maintain activity during the thermal cycling required for PCR.

Summary of The Invention

In a first aspect, the present invention includes a method of detecting a genetic polymorphism in an individual, the method comprising the steps of:

(1) obtaining a sample containing nucleic acid from an individual;

(2) amplifying the nucleic acid sample of step (1) by a method comprising thermocycling and primers, the amplification being carried out in the presence of a thermostable restriction endonuclease which maintains activity during thermocycling, the primers being selected so that they are capable of introducing into nucleic acid amplified from nucleic acid which does not comprise a polymorphism or from nucleic acid which comprises a polymorphism a sequence recognized by the thermostable restriction endonuclease; and

(3) analyzing the product of step (2) to detect the presence or absence of the polymorphism.

In one embodiment of this aspect of the invention, the primer introduces a sequence recognized by the thermostable restriction endonuclease into a nucleic acid amplified from a nucleic acid that does not include the polymorphism.

In a second aspect, the present invention includes a method of detecting a genetic polymorphism in an individual, the method comprising the steps of:

(1) obtaining a sample containing individual nucleic acids;

(2) amplifying the nucleic acid sample of step (1) by a method comprising thermal cycling and primers, the amplification being carried out in the presence of simultaneously active heat-resistant restriction endonucleases, the restriction endonucleases being selected such that they recognize nucleic acids not comprising polymorphisms but not nucleic acids comprising polymorphisms or such that they recognize nucleic acids comprising polymorphisms but not nucleic acids not comprising polymorphisms; and

(3) analyzing the product of step (2) to detect the presence or absence of the polymorphism.

In one embodiment of this aspect of the invention the heat-resistant restriction endonuclease recognizes a nucleic acid that does not include a polymorphism.

In a preferred embodiment of the invention the process further comprises the following additional steps:

(4) reacting the nucleic acid amplified in step (2) with at least one restriction endonuclease selected to digest amplified nucleic acid comprising the particular polymorphism; and

(5) and (3) detecting whether the digestion in the step (4) occurs, wherein the digestion is a mark for the existence of the specific polymorphism.

There are many techniques for amplifying nucleic acids that involve thermal cycling, including Polymerase Chain Reaction (PCR), ligase chain reaction, transcription-based amplification and restriction amplification. However, it is now preferred that the method comprising thermocycling is PCR.

In another preferred embodiment the analysis of step (3) comprises detecting the presence or absence of the amplified nucleic acid of step (2), the presence or absence of the amplified nucleic acid being indicative of the presence or absence of the polymorphism.

Also, the method of the present invention can be applied to various types of nucleic acids, typically DNA.

In another preferred embodiment of the invention the thermostable restriction endonuclease is selected from the group consisting of BstNI, BslI, Tru9I and Tsp 509I.

The methods of the invention may be used to detect a range of genetic polymorphisms, including polymorphisms that occur in one of: ras proto-oncogenes such as K-ras, N-ras and H-ras, or the p53 tumor suppressor gene, or HIV-I, the cystic fibrosis transmembrane conductance regulator, alpha-antitrypsin or beta-globin. The method of the present invention is particularly useful for detecting a polymorphism at codon 12 of K-ras.

The methods of the invention can be used to analyze a range of genetic polymorphisms, including point mutations, small deletions and insertions. It has been found that thermostable restriction endonucleases can be sufficiently thermostable to maintain activity during thermocycling, and that PCR can be performed with various polymerases under the same buffer conditions that maintain restriction endonuclease activity and thermostability. It has been found that inclusion of a thermostable restriction endonuclease in the PCR process results in (i) inhibition of amplification of sequences containing the recognition/cleavage site of the restriction endonuclease and (ii) amplification of only variants lacking the recognition/cleavage site of the restriction endonuclease. These findings allow for the development of a method of restriction endonuclease mediated selective PCR (REMS-PCR). REMS-PCR is simpler than other PCR methods, and utilizes restriction endonucleases to analyze sequence variations. All reaction components are present at the start of the PCR and do not require subsequent manipulation prior to analysis, so the reaction can be completed in a closed vessel or chamber. It has also been found that the inclusion of a heat-resistant restriction endonuclease in the PCR process can result in (i) partial inhibition of nucleic acid amplification containing restriction endonuclease recognition, cleavage sites and (ii) preferential amplification of variants lacking the sequence of restriction endonuclease recognition/cleavage sites.

Detailed description of the invention

As used herein, the following terms and phrases are defined as follows:

PCR is an in vitro DNA amplification method that requires 2 primers flanking the target sequence to be synthesized. A primer is an oligonucleotide sequence that is capable of hybridizing to a target sequence in a sequence-specific manner and extending during PCR. Amplicons (amplics) or PCR products or PCR fragments are extension products that include primers and newly synthesized copies of the target sequence. Multiplex PCR systems contain multiple sets of primers that result in the simultaneous production of multiple amplicons. Primers may be perfectly matched to the target sequence or they may contain internally mismatched bases that result in the introduction of a restriction nuclease recognition/cleavage site in a specific target sequence. Primers may also contain additional sequences and/or modified or labeled nucleotides to facilitate the capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of the primer to its complementary sequence, and annealing of the primer with extension by a DNA polymerase result in exponential amplification of the target sequence. The term target or target sequence refers to the nucleic acid sequence to be amplified. The term template refers to the original nucleic acid to be amplified.

Restriction endonuclease-mediated selective PCR (REMS-PCR) is an analytical method established by the present inventors using the method of the present invention. This method requires both restriction endonuclease and DNA polymerase activity during PCR. Restriction endonucleases suitable for REMS-PCR are preferably active at temperatures compatible with the stringent conditions under which the oligonucleotide primers anneal during PCR, typically 50-65 ℃. A group of commercially available restriction endonucleases with high optimal incubation temperatures in this range is listed in Table 1 below.

The term "subject" is used herein in a broad sense and is intended to encompass human and non-human animals, bacteria, yeast, fungi, and viruses.

TABLE 1

Restriction enzyme	Recognition/cleavage sequences	Optimum incubation temperature
			Acc III	TCCGGA	65℃
AcsI/ApoI	A/G)AATT(T/C)	50℃
			Acy I	G(A/G)CG(C/T)C	50℃
Bco I	C(C/T)CG(A/G)G	65℃
			Bsa BI/Bsi BI	GATNNNNATC	60℃/55℃
Bsa MI	GAATGCN	65℃
			Bsa II	CCNNGG	60℃
Bsa OI	CG(A/G)(T/C)CG	50℃
			Bsa WI	(A/T)CCGG(A/T)	60℃
Bsc BI	GGNNCC	55℃
			Bsc CI	GAATGCN	65℃
Bsc FI	GATC	55℃
			Bse AI	TCCGGA	55℃
Bsi CI	TTCGAA	60℃
			Bsi EI	CG(A/G)(C/T)CG	55℃
Bsi HKAI	G(A/T)GC(A/T)C	65℃
			Bsi LI	CC(A/T)GG	60℃
Bsi MI	TCCGGA	60℃
			Bsi QI	TGATCA	60℃
Bsi WI	CGTACG	55℃
			Bsi XI	ATCGAT	65℃
Bsi ZI	GGNCC	60℃
			Bs/I	CCNNNNNNNGG	55℃
Bsm I	GAATGCN	65℃
			Bsm AI	GTCTCN1/N5	55℃
Bsm BI	CGTCTCN1/N5	55℃
			Bss TII	CC(A/T)(A/T)GG	50℃
Bst I	ACTGGN	65℃
			Bsr DI	GCAATGNN	60℃
Bst TII	GCAGCN8	50℃
			Bst BI	TTCGAA	65℃
Bst NI	CC(A/T)GG	60℃
			Bst UI	CGCG	60℃
Bst YI	(A/G)GATC(C/T)	60℃
			Bst ZI	CGGCCG	50℃
Dsa I	CC(A/G)(C/T)GG	55℃
			Mae II	ACGT	55℃
Mae III	GTNAC	55℃
			Mwo I	GCNNNNNNNGC	60℃
Ssp BI	TGTACA	50℃
			Taq I	TCGA	65℃
Tfi I	GA(A/T)TC	65℃
			Tru 9I	TTAA	65℃
Tsp 45I	GT(C/G)AC	65℃
			Tsp 509I	AATT	65℃
Tsp RI	NNCAGTGNN	65℃
			Tth IIII	GACNNNGTC	65℃

A is adenine, G is guanine, T is thymine, C is cytosine, N is A or G or T or C

In order that the nature of the invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples.

Example 1

Test for evaluating restriction endonuclease Activity/thermostability

The activity/thermostability assay was used to measure the thermostability and residual enzyme activity of restriction endonucleases including BstNI, BslI, Tru9I and Tsp509I in various buffer systems after a certain number of thermal cycles.

The activity/heat resistance of BstNI, BslI and Tru9I was compared in various buffer conditions. A total reaction volume of 25. mu.l contained primers (shown in Table 2 below), dNTPs (dATP, dCTP, dTTP, dGTP) each at 100. mu.M, 0.5 units of Taq DNA polymerase (5 units/. mu.l; AmpliTaq, Perkin Elmer), and either 20 unitsBstNI (10 units/. mu.l; New England Biolabs) or Tru9I (10 units/. mu.l; Boehringer Mannheim) or BslI (50 units/. mu.l; New England Biolabs) at position 10. TABLE 2

Primer and method for producing the same	Amount (pmole)	Present in the following enzyme assay	Sequence of
				5BKIT	7.5	BstNI	TATAAACTTGTGGTAGTTGGACCT
5BKIQ	7.5	BslI，Tru 9I	TATAAACTTGTGGTACCTGGAGC
				3KiE	7.5	BstNI，Bsl I，Tru 9I	CTCATGAAAATGGTCAGAGAAACC
5BKIW	1.25	BslI	TTTTGTCGACGAATATGATCC

In addition, the reaction contained one of the following alkaline buffer systems (listed in table 3), with or without various additional reagents.

TABLE 3

Name of alkaline buffer***New England Biolabs**BoehringerMannheim*Perkin Elmer	Salt (salt)	Tris HCl (pH at 25 ℃ C.)	MgCl2mM	DTTmM
					***NEB2**SuRE/CutM	50mM NaCl	10mM(7.9)	10	1
***NEB3	100mM NaCl	50mM(7.9)	10	1
					*PCR buffer II	50mM KCl	10mM(8.3)
*Stoffel buffer solution	10mM KCl	10mM(8.3)
					MTris 10	50mM NaCl	10mM (8.0 or 8.3 or 8.5 or 8.75)
Htris 50	100mM NaCl	50mM (8.0 or 8.3 or 8.5 or 8.75 or 9.0 or 9.5)

The reaction was placed in a GeneAmp PCR system 9600 thermal cycler (perkin elmer), heated to high temperature and thermally cycled as shown in table 4.

TABLE 4

Restriction endonuclease	BstNI	BslI	Tru9I
				Initial temperature	94 ℃ for 2 minutes	1 minute at 92 DEG C	94 ℃ for 2 minutes
Thermal cycling	60 ℃ for 1 minute and 92 ℃ for 20 seconds	55 ℃ for 1 minute and 92 ℃ for 20 seconds	65 ℃ for 1 minute and 92 ℃ for 20 seconds
				Number of thermal cycles	15 or 30	15 or 30	15 or 30
Optimum temperature for incubation	60℃	55℃	65℃

After thermal cycling, 8. mu.g of plasmid DNA (pGFP-Cl; Clontech) in a volume of 5. mu.l were added to each tube and the reaction was carried out at the optimum temperature listed by the manufacturer for 1 hour. The ability of restriction endonucleases to cleave plasmid DNA was determined by electrophoresis on a 3% NusieeveGTG gel (FMC Bioproducts, Rockland, Md.). The endonuclease is divided into inactivation (I); have low (L), moderate (M) or high (H) activity; or have full (F) activity (table 5).

TABLE 5

Alkaline buffer	Additional reagents	Bst NI Activity		BslI Activity		Tru9I Activity
								Number of cycles		Number of cycles		Number of cycles
		15	30	15	30	15	30
								NEB2SuRE Cut M		M	L			M	L
NEB3		F	M
								1 XPCR buffer II	3 mM MgCl2	M	L
6 mM MgCl2	M	L	I	I
							10 mM MgCl2		H	M	I	I
10 mM MgCl2·1mM DTT			H	M
							1 × Stoffel buffer solution		3 mM MgCl2	M	L
6 mM MgCl2	M	L	I	I
								10 mM MgCl2	M	L	I	I
10 mM MgCl2·1mM DTT			H	M
								MTris 10 pH 8.3	10 mM MgCl2	H	L	I	I
HTris 50 pH 8.3	F	M
							MTris 10 pH 8.0	10 mM MgCl2·1mM DTT
MTris 10 pH 8.3	10 mM MgCl2·1mM DTT			H	M
							MTris 10 pH 8.0	10 mM MgCl2		M	L
MTris 10 pH 8.3	H	L	I	I
							MTris 10 pH 8.5		M	L	I	I
MTris 10 pH 8.75			I	I
							10 mM MgCl2·1mM DTT				H	M	H	L
	HTris 50 pH 8.5HTris 50 pH 8.5	6 mM MgCl2			I	I
6 mM MgCl2·1mM DTT										H	M
		HTris 50 pH 8.0	10 mM MgCl2	F	M
HTris 50 pH 8.3	F						M		I	I
		HTris 50 pH 8.5HTris 50 pH 8.5		F	M	I		I
10 mM MgCl2·1mM DTT									H	M	H	L
			HTris 50 pH 8.75	10 mM MgCl2				I					I
10 mM MgCl2·1mM DTT							H		M	H	L
				HTris 50 pH 8.3	3 mM MgCl2	H		M
6 mM MgCl2	F	M
					10 mM MgCl2	F		M			I	I
HTris 50 pH 8.3	10 mM MgCl2·1mM DTT						H		M
				HTris 50 pH 8.36mMMgCl2	-	F		M
1 mM DTT	H	M
					0.1mg/ml acetylated BSA (aBSA)	H		M
0.1mg/ml non-acetylated BSA (non-a BSA)	H	M
					1 mMDTT+aBSA	M		M
1 mM DTT++non-aBSA	M	L
					10% Glycerol	H		M
HTris 550 pH 9.0	10mMMgCl2·1mM DTT									H					M
				HTris 50 pH 9.5	10mMMgCl2·1mM DTT								F	M

These experiments show that the activity/thermostability of restriction endonucleases during thermocycling varies greatly depending on the restriction endonuclease and the buffer system in which it is placed. pH and ionic Strength in Tris buffer, monovalent cation (K)⁺Or Na⁺) Selection and concentration of free Mg²⁺The concentration of (a) and the presence of other additives, especially DTT, affect the activity/heat resistance. The effect of each of these components will depend on the other components in the buffer, e.g., BstNI in the presence of 10mM MgCl₂The PCR buffer solution II contains 3 or 6mM MgCl₂More activity was retained in the buffer of (1). In contrast, MgCl was varied between 3-10 mM when in HTris 50(pH8.3) buffer₂The concentration of (d) had little effect on Bst NI activity. In another example, buffer pH had a greater effect on Bst NI thermostability/activity in MTris 10 than HTris 50.

Bstni remained fully active after 15 thermal cycles and moderately active after 30 thermal cycles in a buffer system containing one of the following: i)100mM NaCl, 50mM Tris HCl (pH8.3) and 6mM MgCl₂Or ii)100mM NaCl, 50mM TrisHCl (pH8.0-8.5) and 10mM MgCl₂Or iii) NEB3 buffer and 0.1 mg/ml. Bst NI was more active in these buffers during thermal cycling than NEB2 buffer with 0.1mg/ml acetylated BSA with manufacturer's recommended buffer conditions.

Similar assay Bsl I activity/thermostability experiments showed that the presence of 1mM DTT was required to maintain the endonuclease activity after thermal cycling. If DTT is present, BslI retains activity over a wide range of conditions. BslI maintained moderate activity after 30 cycles of thermal cycling in a buffer system containing: i) PCR buffer II(Perkin Elmer), 1mM DTT and 10mM MgCl₂Ii) Stoffel buffer (Perkin Elmer), 1mM DTT and 10mM MgCl₂Iii)50mM NaCl, 10mM Tris HCl (pH8.5), 1mM DTT and 10mM MgCl₂Iv)100mM NaCl, 50mM TrisHCl (pH8.3-8.5), 1mM DTT and 10mM MgCl₂Or v)100mM NaCl, 50mM TrisHCl (pH8.5), 1mM DTT and 6mM MgCl₂. Tru9I was cultured in the presence of 100mM NaCl, 50mM TrisHCl (pH8.5-9.25), 10mM MgCl₂And 1mM DTT after 30 thermal cycles maintained moderate activity. Similar experiments as described above showed that Tsp509I contained 50mM NaCl, 10mM TrisHCl (pH 9.0-10), 10mM MgCl₂And 1mM DTT maintained moderate activity after 30 cycles of thermal cycling.

Example 2

Identification of buffer systems compatible with restriction endonuclease and DNA polymerase thermotolerance/activity and PCR

Determination of buffer range that can be used to maintain BstNI resistance/activity (above) also assessed its compatibility with PCR using primers 5BKIT or 5BKIW and 3 KiE. A PCR mixture containing 100. mu.M each of genomic K562DNA (800ng), 30pmol 5BKIT or 30pmol 5BKIW, 30pmol 3KiE, and dNTPs (dATP, dCTP, dTTP, dGTP) was established for each buffer system. 4 units of Taq DNA polymerase (5 units/. mu.l; AmpliTaq, Perkin Elmer) and Taq Start^TMAntibodies (0.16. mu.l in 3.8. mu.l antibody dilution buffer; Clontech) were mixed to generate Taq DNA polymerase in a final molar ratio of 1: 5: TaqStart^TMAn antibody. Taq DNA polymerase: taq Start^TMThe antibody mixture was incubated at room temperature for 15 minutes before adding the above mixture. The total reaction volume was 100. mu.l. The reaction was placed in a GeneAmp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 2 minutes and then cycled through 30 cycles of 60 ℃ for 1 minute and 92 ℃ for 20 seconds. The reaction was maintained at 60 ℃ for 15 minutes after thermal cycling.

Without subsequent manipulation, 28. mu.l aliquots of each reaction were analyzed by electrophoresis on a 5% Nusieve GTG gel (FMCBioproducts, Rockland, Md.) photographed using the Stratagene Eagle Eye II Video system. The amplification efficiency of primers 5BKIT and 3KiE, or 5BKIW and 3KiE were determined as low, medium or high. These primers were designed for use in a multiplex REMS-PCR system in combination with the restriction endonuclease bstni. The analysis of Bst NI activity/heat resistance and PCR were done in the same reaction buffer and subjected to the same thermal cycling procedure. The results of both assays were examined to find conditions that allowed both efficient PCR amplification and maintained restriction endonuclease activity (table 6).

TABLE 6

Alkaline buffer	Additional reagents	Efficiency of PCR amplification		BstNI Activity
						5BKIT3KiE	5BKIW3KiE	15 cycles	30 cycles
		NEB2		Height of	Height of					Medium and high grade	Is low in
NEB3						Height of	Medium and high grade	Is totally produced from	Medium and high grade
		PCR buffer II	3mM MgCl2	Height of	Height of					Medium and high grade	Is low in
6mM MgCl2	Height of					Height of	Medium and high grade	Is low in
			10mM MgCl2	Height of	Height of				Height of	Medium and high grade
Stoffel buffer solution	3mM MgCl2					Height of	Height of	Medium and high grade			Is low in
		6mM MgCl2	Height of	Height of	Medium and high grade				Is low in
	10mM MgCl2					Medium and high grade	Medium and high grade	Medium and high grade		Is low in
		MTris 10 pH8.3	10mM MgCl2	Height of	Height of				Height of		Is low in
HTris 50 pH8.3	Medium and high grade					Medium and high grade	Is totally produced from	Medium and high grade
		MTris 10 pH8.0		10mM MgCl2	Height of				Medium and high grade	Medium and high grade	Is low in
MTris 10 pH8.3	Height of		Height of			Height of	Is low in
		MTris 10 pH8.5			Height of			Medium and high grade	Medium and high grade	Is low in
HTris 50 pH8.0	10mM MgCl2		Medium and high grade			Medium and high grade	Is totally produced from				Medium and high grade
		HTris 50 pH8.3		Medium and high grade	Medium and high grade			Is totally produced from	Medium and high grade
HTris 50 pH8.5			Is low in			Medium and high grade	Is totally produced from			Medium and high grade
		HTris 50 pH8.3		3mM MgCl2	Height of			Medium and high grade	Height of		Medium and high grade
6mM MgCl2	Height of		Medium and high grade			Is totally produced from	Medium and high grade
				0mM MgCl2	Medium and high grade			Medium and high grade	Is totally produced from	Medium and high grade
HTris 50 pH8.36mM MgCl2	-		Height of			Medium and high grade	Is totally produced from				Medium and high grade
		DTT		Height of	Medium and high grade			Height of	Medium and high grade
	aBSA		Height of			Medium and high grade	Height of			Medium and high grade
		non-aBSA		Height of	Medium and high grade			Height of	Medium and high grade
	DTT+aBSA		Height of			Medium and high grade	Medium and high grade			Medium and high grade
		DTT + non-aBSA		Height of	Medium and high grade			Medium and high grade	Is low in
	Glycerol		Is low in			Is low in	Height of			Medium and high grade
		T4 gene 32 protein		Height of	Is low in			Is low in	Deactivation of the enzyme

Buffer conditions were selected that resulted in both i) high efficiency amplification with primer pair 5BKIT and 3KiE and moderate efficiency amplification with primer pair 5BKIT w and 3KiE and ii) retention of full Bst NI activity after at least 15 thermal cycles and moderate activity after 30 thermal cycles for REMS-PCR assays that required both DNA Taq polymerase and Bst NI activity. Buffer conditions suitable for these standards were 100mM NaCl, 50mM TrisHCl pH8.3 and 6mM MgCl₂。

Example 3

REMS-PCR using Bst NI and DNA Taq polymerase: analysis of K-ras gene codon 12 in multiplex system with addition of internal control.

The REMS-PCR method was used to detect point mutations at codon 12 of the K-ras oncogene. The human cell lines Calu I [ ATTC HTB54] and K562[ ATCC CCL243] were obtained from the American type culture Collection, Calu I being a lung adenocarcinoma cell heterozygous at K-ras codon 12 with both wild type (GGT) and mutant (TGT) sequences (D.J.Capon1983 Nature 304, 507- > 513), K562 being a human leukemia cell line with K-ras codon 12 being wild type (R.L.Ward et al, molecular Pathology 1995, 48, M273-277). Genomic DNA was extracted from CaluI and K562 using standard techniques (Sambrook et al 1989). DNA samples were amplified by REMS-PCR using primers 5BKIT, 5BKIW, 3MKiC and 3KiE (Table 7).

TABLE 7

Guiding article	Function of	Sequence of
			5BKIT	Diagnostic primers	TATAAACTTGTGGTAGTTGGACCT
5BKIW	PCR control primer	TTTTGTCGACGAATATGATCC
			3MKiC	BstNI control primer	CTGTATCAAAGCTTGGTCCTGGACCAG
3KiE	3' primer	CTCATGAAAATGGTCAGAGAAAC

The bolded C in primer 5BKIT is a mismatch with respect to the K-ras gene sequence, and if codon 12 is wild-type, the mismatched base results in the introduction of a BstM recognition/cleavage site in the K-ras amplicon. Amplicons containing a mutation at nucleotide 1 or 2 of codon 12 did not contain a Bst M recognition/cleavage sequence. Primers 5BKIT and 5BKIW were labeled with biotin at their 5' ends and produced similarly labeled PCR amplicons. The bold G of primer 3MKiC is a mismatch with respect to the K-ras sequence, which results in the introduction of a BstM recognition/cleavage site inside the primer and will be incorporated into any amplicon generated by amplification with this primer and either 5BKIT or 5 BKIW.

Genomic DNA of K562, Calu I and Calu I: a1: 10 mixture (weight ratio) of K562 was amplified in a multiplex REMS-PCR system containing 100mM NaCl, 50mM Tris (pH8.3) and 6mM MgCl₂The genomic DNA of (800ng), 30pmol 5BKIT, 30pmol 3KiE, 5pmol 5BKIW, 80pmol3MKiC, dNTP (dATP, dCTP, dTTP, dGTP) each 100. mu.M, 80 units of Bst NI (10 units/. mu.l, New England Biolabs) and 4 units of Taq DNA polymerase (5 units/. mu.l, Ampli Taq, Perkin Elmer). The total reaction volume was 100. mu.l, and the two control reactions contained either CaluI DNA or dH₂O (no DNA) and no Bst NI was present. The reaction was placed in the Gene Amp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 3 minutes, then subjected to 30 cycles of 60 ℃ for 1 minute followed by 92 ℃ for 20 seconds, and then thermally cycled and maintained at 60 ℃ for 15 minutes.

Mu.l of the product of each reaction was analyzed without subsequent manipulation by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rockland, Md.), which was photographed with a Polaroid Land camera. In the control reaction containing CaluI DNA but no BstNI, three fragments were clearly visible; a 185bp fragment including the amplicon incorporating primers 5BKIT and 3KiE, a 156bp fragment including the amplicon incorporating primers 5BKIT and 3MKiC, and a 114bp fragment including the amplicon incorporating primers 5BKIW and 3 KiE. A fragment of 85bp including the amplicon incorporating the primers 5BKIW and 3MKiC was weakly visible.

In reactions containing Bst NI, the presence of a 185bp fragment, which can be seen in a reaction containing Calu I and CaluI in a 1: 10 ratio: in the reactions with K562DNA, but not in the reactions with K562DNA alone, the Bst NI control fragment of 156bp (and 85bp) was not visible in any of the reactions with Bst NI, demonstrating that Bst NI mediates complete inhibition of amplification of the second fragment. Since any 156bp amplicon will contain a Bst NI site, inhibition of amplification of this fragment is independent of the mutation status of codon 12. The absence of the restriction endonuclease control fragment clearly indicates a negative result. A114 bp PCR control fragment was found in all reactions including the K562-containing DNA reaction, confirming that the reaction conditions, including the amount of template DNA, were sufficient for amplification by PCR. The presence of the PCR control fragment clearly judged a positive result. No fragments were found in the reaction without DNA.

Example 4

REMS-PCR: limits of detection of Point mutations

The limit of point mutations detected by REMS-PCR was assessed by analyzing the codon 12 of the K-ras gene in samples containing Calu I DNA diluted with Sup TI DNA. Sup TI [ ATCC CRL 1942]Is a leukemia cell line obtained from the American type culture Collection. Calu I is a heterozygous mutant for K-ras codon 12 and Sup TI is a wild type for K-ras codon 12. Genomic DNA was extracted from these cell lines using standard techniques (Sambrook et al 1989) and amplified by REMS-PCR. CaluI DNA was diluted 1: 10, 1: 10 with Sup TIDNA²、1∶10³、1∶10⁴、1∶10⁵And 1: 10⁶Ratio of Calu I: Sup T1 (weight ratio).

The REMS-PCR reaction contained 100mM NaCl, 50mM Tris (pH8.3), and 6mM MgCl₂Genomic DNA (1. mu.g), 30pmole 5BKIT, 30pmole 3KiE, 5pmole 5BKIW, dNTPs (dATP, dCTP, dTTP, dGTP) each 100mM, and 40 units of Bst NI (10 units/. mu.l, New England Biolabs). 4 units of Taq DNA polymerase (5 units/. mu.l; AmpliTaq, Perkin Elmer) and TaqStart^TM(0.16. mu.l in 3.8. mu.l of antibody dilution buffer; Clontech) to generate Taq DNA polymerase in a final molar ratio of 1: 5: TaqStart^TM。TaqDNA polymerase: taq Start^TMThe antibody mixture was incubated at room temperature for 15 minutes before adding the PCR mixture. The total reaction volume was 100. mu.l. The reaction was placed in the Gene Amp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 2 minutes followed by 30 cycles of 60 ℃ for 1 minute followed by 92 ℃ for 20 seconds. The reaction was maintained at 60 ℃ for 15 minutes after thermal cycling.

A28. mu.l aliquot of each reaction was analyzed without subsequent manipulation by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rockland, Md.), which was photographed using a Polaroid land (Baoli.) camera and Stratagene eagle eye II imaging system. A185 bp fragment generated by amplification with primers 5BKIT and 3KiE, which was visualized by Polaroid photography and eagle eye imaging as containing 1: 10, was used to diagnose the presence of the codon 12 mutation²And 1: 10³The reaction of the ratio of Calu I: Sup T1DNA and the results of eagle eye imaging can be seen to contain 1: 10⁴In the reaction of the ratio. The 185bp fragment was neither found to contain 1: 10⁵And 1: 10⁶The reaction with the ratio of Calu I to Sup T1DNA was also not seen in the reaction with Sup T1 alone. The 114bp PCR control fragment generated from amplification with the primer 5BKIW and 3KiE fragments was found in all reactions, demonstrating that the reaction conditions, including the amount of template DNA, were sufficient to be efficiently amplified by PCR.

The REMS-PCR reaction was also subjected to a colorimetric assay similar to that described by Findlay et al (clinical chemistry, 199339/9, 1927-. The PCR amplicons are specifically captured by hybridization to oligonucleotide probes covalently attached to latex beads applied to separate locations of a Periodotal Surecell blank. The sequence of the capture oligonucleotides and the list of specific PCR amplicons captured are as follows (table 8). K-Cap and K-Cap2 were specifically designed to capture only the diagnostic K-ras amplicon generated by amplification with primers 5BKIT and 3KiE via the mutant template. K-Cap3 was designed to capture amplicons generated from amplification of either mutant or wild-type templates with 5BKIT or 5BKIW and 3 KiE. H-Capl captures non-specific amplicons and provides a negative control for non-specific amplification or hybridization.

TABLE 8

Probe (function)	Sequence of	Homologous fragment size (incorporated primer)	Captured amplicon types
				K-Capl (diagnostic)	TAGCTGTATCGTCAAGGCACTCTT	185bp(5BKIT/3KiE)	Mutant only
K-Cap2 (diagnostic)	AAATGATTCTGAATTAGCTGTATCGTC	185bp(5BKIT/3KiE)	Mutant only
				K-Cap3(PCR control)	GCACCAGTAATATGCATATTAAAACAAG	185bp(5BKIT/3KiE)114bp(5BKIW/3KiE)	Mutant wild type
H-Capl (negative control)	ACCATCCAGCTGATCCAGAACCAT	Nil	Non-specificity

Aliquots of 4 oligonucleotide latex beads (dispersed at 0.25% in 1.6. mu.l 10mM Tris, 1mM EDTA pH 7.4) were applied to different sites on the Surecell membrane, with all 4 oligonucleotides located in each Surecell well. The oligonucleotide latex beads were allowed to dry for 15 minutes. 30 μ l aliquots of each PCR were mixed with 170 μ l50mM KCl, 10mM Tris (pH8.3) and 10mM MgCl₂And (6) diluting. The solution was denatured at 95 ℃ for 6 minutes and applied to Surecell wells. Surecells was then incubated at 50 ℃ for 5 minutes to allow hybridization of the PCR amplicons to the capture oligonucleotides. With 300. mu.l of 50mM KCl, 10mM Tris (pH8.3) and 10mM MgCl₂The wells were washed at 50 ℃. The hybridized amplicons were reacted with three drops of streptavidin conjugate bound to horseradish peroxidase (EC1.11.1.7) and incubated for 2 minutes at room temperature. The washing step was repeated to minimize non-specific reactions. 4 drops of Leucodye/H were added₂O₂And Surecell was incubated at room temperature for 2 minutes. The immobilized complex acts as a catalyst in the oxidative conversion of the dye molecules from the colorless to the blue form. The reaction was performed with 4 drops of 0.1% NaN₃And (6) terminating. The resulting colored spots were visually scored by comparison with a color chart and graded from 0 (colorless) to 10 (dark blue) (Table 9)

TABLE 9

Color score
								CaluI：SupTIDNA	1∶10	1∶102	1∶103	1∶104	1∶105	1∶106	SupTl
K-Cap 1 (mutant-specific)	9	8	4	2	0	0	0
								K-Cap2 (mutant specific)	9	8	4	2	0	0	0
K-Cap3(PCR control)	9	9	9	9	9	9	9
								H-Capl (non-specific negative control)	0	0	0	0	0	0	0

The sensitivity of the REMS-PCR method when analyzed by gel electrophoresis or colorimetry is present at 1: 10³～∶1∶10⁴When in the wild type sequence background allows for the selective amplification of K-ras codon 12 mutation sequence detection. The wild-type K-ras codon 12 sequence was not detected in this REMS-PCR analysis. The literature indicates that this level of sensitivity will be sufficient to analyze DNA extracted from clinical specimens, including tissue resection and biopsy, cytological samples and body fluids/secretions such as feces, urine and sputum containing small amounts of exfoliated tumor cells.

In the clinic, where large numbers of samples are analyzed simultaneously, it is necessary that amplification does not begin in advance, as this can lead to amplification of non-specific products, including primer dimers. The monoclonal antibody will bind to DNA Taq polymerase, thus inhibiting activity and amplification prior to the first denaturation step. In the initial experiments with REMS-PCR, 1: 28 DNA Taq polymerase recommended by Clontech: taq Start^TMDue to amplification of the wild-type Sup Tl DNA template, resulting in false positive results, various molar ratios were examined and DNA Taq polymerase was established: taq Start^TMA low molar ratio of antibody, such as 1: 5, inhibits non-specific amplification and primer dimer formation without false positive results.

EXAMPLE 5 analysis of clinical specimens by REMS-PCR

Genomic DNA was extracted from normal colonic mucosa (NC) and Colon Adenocarcinoma (CA) using standard procedures (Sambrook et al 1989). The presence of K-ras codon 12 mutation in the sample was analyzed by REMS-PCR with the following changes in the procedure of example 4; DNA (0.5. mu.g) was amplified in the presence of 4 units Taq DNA polymerase and 80 units Bst NI. Aliquots of 30 μ l of each reaction were analyzed by electrophoresis on 5% Nusieve GTG gels (FMC Bioproducts, Rockland, Md.) without subsequent manipulation.

The 185bp fragment generated by amplification with primers 5BKIT and 3KiE was diagnosed for the presence of a mutation in codon 12 and was visualized by gel electrophoresis in two reactions containing DNA from adenocarcinoma samples CA7 and CAg, the diagnostic fragment was not visualized in the other two reactions containing DNA from adenocarcinoma samples CA1 and CA2, nor in the 4 reactions containing DNA extracted from normal colon mucosa NC1, NC2, NC7 and NC 8. The 114bp control fragment generated by amplification with primers 5BKIW and 3KiE was seen in all reactions, indicating that efficient PCR amplification occurred in all reactions.

Genomic DNA of colonic tissue the presence of K-ras codon 12 mutation was previously analyzed by standard enrichment PCR (R.L Ward et al, molecular Pathology 199548, M273-277). Amplification by REMS-PCR or enrichment PCR followed by gel electrophoresis analysis gave consistent results. Both PCR methods indicated that the DNA of adenocarcinoma samples CA7 and CA8 harbored a mutation at codon 12 of K-ras while the DNA of adenocarcinoma CA1 and CA2, as well as normal mucosal samples NC1, NC2, NC3, and NC4 were wild-type at codon 12 position. These results demonstrate that REMS-PCR is suitable for rapid analysis of clinical specimens. Example 6: REMS-PCR: system allowing identification of specific nucleotide substitutions

The REMS-PCR system was used to detect point mutations at codon 12 of the K-ras oncogene, and additional analysis with restriction endonucleases confirmed both the diagnosis of the codon 12 mutation and allowed the identification of specific nucleotide substitutions. The human cell lines CaluI [ ATCC HTB54], A549[ ATCC ], K562[ ATCC CCL243], Sup T1[ ATCC CRL 1942] were obtained from the American type culture Collection. Calu I is a lung adenocarcinoma cell heterozygous at K-ras codon 12 with both wild type (GGT) and mutant (TGT) sequences (D.J.Capon1983 Nature 304, 507-513). A549 is a lung adenocarcinoma cell with K-ras codon 12 homozygous mutant (AGT) (D.M. Vallenzuela and J.Groffen1986 NAR 14, 843-852). K562 and Sup T1 are leukemia cell lines with K-ras codon 12 being wild type. Genomic DNA was extracted from these cell lines using standard techniques (Sambrook et al 1989).

REMS-PCR was performed with primers 5BKIT and 3AKIP, which simultaneously introduce multiple restriction endonuclease recognition/cleavage sites. Primers 5BK5 and 3K6 acted as PCR control primers. (watch 10)

Watch 10

Primer and method for producing the same	The sequence is as follows: the bases mismatched with the K-ras gene introduced into the restriction enzyme site are shown in bold (additional mismatched bases are underlined)
		5BKIT	TATAAACTTGTGGTAGTTGGACCT
3AKIP	G GA TGAC TCA TTAAGGCACTCTTGCCTACGCCC
		5BK5	TCAGCAAAGACAAGACAGGTA
3K6	AGCAATGCCCTCTCAAGA

Primer 5BKIT resulted in the introduction of a Bst NI recognition/cleavage site in the K-ras amplicon with codon 12 wild type. Primer 3AKIP induced recognition/cleavage sites of one or more restriction enzymes BsaJI, StyI, AvrII, MnlI, AciI, RleI and Bsu36I in the codon 12 mutated K-ras amplicon, as shown below (Table 11). TABLE 11

The mismatched bases introduced by the K-ras sequence and the induced restriction enzyme recognition/cleavage sites (5 BKIT (C)) and 3AKIP (G)) are shown in bold, the point mutation at codon 12 is shown by underlining, N ═ T or A or C or G)
						Codon 11	Codon 12	Codon 13	Restriction enzyme
Wild type sequence	CCTCCT	GGGGG	GGC	Bst NI
					Mutant sequences	CCN	NGG		Bsa JI
CCT	TGG		Sty I
				CCT		AGG		AvrII/Sty I
CCT	C		Mnl I
						G CG	G	Aci I
T	G TG	GG	Rle AI
				CCT		N AGG AG	GG	Bsu 36IMnl I

The expected pattern of cleavage sensitivity and resistance of the mutant amplicons to restriction endonucleases Bsa JI, StyI, AvrII, MnlI, AciI, RleI and Bsu36I is dependent on the actual mutation at codon 12 and is listed in Table 12. TABLE 12

Codons 12 positions 1 and 2 (point mutations underlined, N ═ T or a or C)	Restriction endonucleases for cleaving mutant amplicons	Restriction endonucleases that do not cleave mutant amplicons
			NG	Bsa JI
TG	Bsa JI/Sty I	Avr II
			AG	Bsa JI/Sty I/Avr II
CG	Bsa JI/Mnl I	Bsu 36I
			GN		Bsa JI
GT	Rle AI	Bsa JI
			GC	Aci I	Bsa JI
GA	Mnl I/Bsu 36I	Bsa JI

Genomic DNA of the human cell lines CaluI, A549, K562 and SupTl was amplified in a multiplex REMS-PCR system in 100mM NaCl, 50mM Tris (pH8.3) and 6mM MgCl₂The DNA contains genomic DNA (500ng), 50pmole 5BKIT, 50pmole3AKIP, 3pmole 5BK5, 3pmole 3K6, dNTP (dATP, dCTP, dTTP, dGTP) each at 100mM, 40 units of Bst NI (10 units/. mu.l, New England Biolabs). 4 units of Taq DNA polymerase (5 units/. mu.l; Ampli Taq, amber)Gold elmer) and Taq Stat^TMAntibodies (0.06. mu.l in 1.5. mu.l antibody dilution buffer, Clontech) were mixed to generate Taq DNA polymerase in a final molar ratio of 1: 2: taq Start^TMAn antibody. Taq DNA polymerase was added to the reaction: taq Start^TMThe antibody mixture was incubated at room temperature for 15 minutes. The total reaction volume was 100. mu.l. The reaction was placed in a Gene Amp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 3 minutes and then subjected to 30 cycles of 60 ℃ for 1 minute followed by 92 ℃ for 20 seconds. The reaction was maintained at 60 ℃ for 15 minutes after thermal cycling.

Aliquots of 20. mu.l of each reaction were analyzed without subsequent manipulation by electrophoresis on 5% Nusieve GTG gels (FMC Bioproducts, Rockland, Md.) photographed with a Polaroid camera. A58 bp fragment generated by amplification with primers 5BKIT and 3AKIP, which was found in reactions containing Calu I and A549DNA but not SupTl or K562DNA, was diagnostic for the presence of the codon 12 mutation. The 167bp PCR control fragment generated by amplification with primers 5BK5 and 3K6 was present in all reactions, including those containing SupTl and K562 DNA. This confirms that efficient PCR amplification occurred in all reactions.

A15. mu.l aliquot containing the CaluI or A549DNA reaction was digested with 10 units of restriction endonucleases BsaJI, StyI, AvrII, MnlI or AciI (as shown in Table 13 below) and incubated at the optimal digestion temperature as specified by the manufacturer (New England Biolabs). The reaction was analyzed by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rockland, Md.) and the gel was photographed with a Paoli camera. Watch 13

Template DNA	Primers for generating amplicons	Restriction endonuclease	Results	Codon 12 sequences at positions 1 and 2 (N ═ A, C or T)
					K562	Only 5K5/3K6	-	-	Wild type-GG
SupTl	Only 5K5/3K6	-	-	Wild type-GG
					CaluI	5BKIT/3AKIP5K5/3K6	Bsa JISty IAvr II	Resistance to cleavage	Mutant NGTG or AG non-AG outcome: mutant (TG)
A549	5BKIT/3AKIP5K5/3K6	Bsa JISty IAvr II	Cutting and cutting	Mutant NGAG or TGAG results: mutant (AG)

This REMS-PCR system allows the detection of codon 12 mutations in the K-ras oncogene. Subsequent restriction endonuclease analysis confirmed the presence of the mutation and allowed the identification of specific nucleotide substitutions.

Example 7: REMS-PCR System Using Bst NI and Stoffel polymerase

Human cell line Calu I [ ATCC HTB54]And SupT1[ ATCC CRL 1942]The genomic DNA of (3) was amplified by REMS-PCR. Genomic DNA was extracted from these cell lines using standard techniques (Sambrook et al 1989) and amplified by REMS-PCR in 10mM KCl, 10mM Tris (pH8.3) and 10mM MgCl₂Genomic DNA (1. mu.g) from (1 XStoffee buffer; Perkin Elmer), 30pmol of 5BKIT, 30pmol of 3KiE, 2pmol of 5BKIW, dNTPs (dATP, dCTP, dTTP, dGTP), 100mM each, and 40 units of Bst NI (10 units/. mu.l, New England Biolabs), control reaction DNA-free (dH)₂O). 5 units of Stoffel fragment (10 units/. mu.l; Perkin Elmer) were mixed with Taq antibody TP4(D.J.Sharkey et al 1994 Bio/technology 12, 506-509) (0.05. mu.l in 1.2. mu.l Clontech antibody dilution buffer) to produce a final molar ratio of 1: 2 of Stoffel fragment: taq antibody TP 4. Stoffel fragment was added to the reaction before: the Taq antibody mixture was incubated at room temperature for 15 minutes. The total reaction volume was 100 ul. The reaction was placed in a Gene Amp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 2 minutes and then subjected to 30 cycles of 60 ℃ for 1 minute followed by 92 ℃ for 20 seconds. The reaction was maintained at 60 ℃ for 15 minutes after thermal cycling.

A25. mu.l aliquot of each reaction was analyzed by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rockland, Md.) photographed by a Paoli camera without subsequent manipulation. A185 bp fragment generated by amplification with primers 5BKIT and 3KiE, which was found in the reactions with Calu I DNA but not Sup TI DNA, was diagnostic for the presence of the codon 12 mutation, and a 114bp PCR control fragment generated by amplification with primers 5BKIW and 3KiE, which was found in both reactions, indicating efficient PCR amplification. No fragments were visible in the control reaction without template.

Example 8: REMS-PCR System Using Bsl I and Taq DNA polymerase

REMS-PCR analysis was developed to detect point mutations at codon 12 of the K-ras oncogene. In this analysis, if the amplicons are wild-type at codon 12, they contain the recognition/cleavage sequence of the thermostable restriction endonuclease Bsl I. Amplicons containing mutations at the first or second nucleotide of codon 12 do not contain the recognition/cleavage sequence of Bsl I.

Human cell line CaluI [ ATCC HTB54]And K562[ ATCC CCL243]]The genomic DNA of (3) was amplified by REMS-PCR. Calu I is a heterozygous mutant at codon 12 of the K-ras gene and K562 is a wild type at codon 12. Genomic DNA was extracted from these cell lines using standard techniques (Sambrook et al 1989) and Calu I DNA was diluted 1: 10, 1: 10 with K562DNA²、1∶10³Calu I of (1): k562 ratio (weight).

DNA was amplified by REMS-PCR using primers 5BKIQ, 5BKIW, and 3KiH (Table 14). The 2 bold C's in 5BKIQ are mismatches with respect to the sequence of the K-ras gene, and these mismatched bases result in the introduction of a Bsl I site in an amplicon in which codon 12 is wild-type. Primers 5BKIQ and 5BKIW were biotinylated.

TABLE 14

Primer and method for producing the same	Sequence of
		5BKIQ	TATAAACTTGTGGTACCTGGAGC
5BKIW	TTTTGTCGACGAATATGATCC
		3KiH	GAAAATGGTCAGAGAAACC

The reaction contained solutions in 100mM NaCl, 50mM Tris (pH8.5), 1mM DDT and 6mM MgCl₂400ng of genomic DNA, 30pmole 5BKIQ,15pmole3KiH, 0.5pmole 5BKIW, dNTPs (dATP, dCTP, dTTP, dGTP), 100. mu.M each and 10 units of Bsl I (50 units/. mu.l, New England Biolabs). 8 units of Taq DNA polymerase (5 units/. mu.l; Ampli Taq, Perkin Elmer) and Taq Start^TMAntibodies (0.16. mu.l in 3.8. mu.l antibody dilution buffer; Clontech) were mixed to generate Taq DNA polymerase in a final molar ratio of 1: 5: taq Start^TMAn antibody. Taq DNA polymerase: taq Start^TMThe antibody mixture was incubated at room temperature for 15 minutes before addition to the reaction. The total reaction volume was 50. mu.l. The reaction was placed in a Gene Amp PCR system 9600 (Perkin Elmer) and denatured at 94 ℃ for 2 min. The reaction was then subjected to 10 cycles of 63 ℃ for 30 seconds followed by 92 ℃ for 20 seconds and then 20 cycles of 55 ℃ for 1 minute followed by 92 ℃ for 20 seconds. The reaction was maintained at 55 ℃ for 15 minutes after thermal cycling.

A28. mu.l aliquot of each reaction was analyzed by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rock/and, MD) photographed by Baoli camera without subsequent manipulation. The presence of the codon 12 mutation was diagnosed by a 180bp fragment generated by amplification with primers 5BKIQ and 3KiH, which fragment was found to contain 1: 10 and 1: 10²In the reaction of Calu I: K562. The 180bp diagnostic fragment could not be found in the 1: 10 DNA fragment³In the reaction with the ratio Calu I: Sup T1 or with K562 only. The 109bp PCR control fragment generated by amplification with primers 5BKIW and 3KiH was found in all reactions indicating efficient amplification by PCR.

This system uses restriction endonuclease Bsl I to detect mutations at K-ras codon 12, which can be used to detect the system of mutations at any one of codons 12 or 13 of the 3 ras-oncogenes K-ras, H-ras and N-ras, and also to analyze other mutations occurring at codons encoding glycine or proline or other mutations occurring at nucleotides C or G.

Example 9: analysis of K-ras codon 12 by REMS-PCR method requiring subsequent BstNI digestion

An alternative method for detecting K-ras oncogenePoint mutation of codon 12. From Calu I [ ATCC HTB54 using standard techniques (Sambrook et al 1989)]And K562[ ATCC CCL243]]Extracting genome DNA. Calu I DNA was obtained using K562DNA at 1: 10, 1: 10²、1∶10³And 1: 10⁴Diluted with Calu I: K562 (by weight). Primer 5BKIM with sequence GACTGAATATAAACTTGTGGTAGTTGGACCT and DNA sample with sequenceGG ATG ACTCAT TTTCGTCCACAAAATGATTCTGAATTAG primer 3 AKIL. Bold C in primer 5BKIM are mismatches with respect to the K-ras gene sequence and result in the introduction of a recognition/cleavage site for Bst NI in the K-ras amplicon if they are wild-type at codon 12. The 3AKIL and K-ras mismatched bases are underlined.

A reaction volume of 100. mu.l contained genomic DNA (800ng), 40pmole 5BKIM, 40pmol 3AKIL, 100. mu.M each of dNTPs (dATP, dCTP, dTTP, dGTP), 10. mu.l of 10 XPCR buffer II (Perkin Elmer), 1.5mM MgCl₂80 units of Bst NI (10 units/. mu.l, New England Biolabs) and 2 units of Taq DNA polymerase (5 units/. mu.l; Ampli Taq, Perkin Elmer). The reaction was placed in a Gene Amp PCR system 9600 (Perkin Elmer), denatured at 94 ℃ for 3 minutes and then subjected to 40 cycles of 1 minute at 60 ℃ followed by 20 seconds at 92 ℃. The reaction was maintained at 60 ℃ for 15 minutes after thermal cycling.

Aliquots of 25. mu.l of each reaction were not analyzed by subsequent manipulations. A second 25. mu.l aliquot of each reaction was incubated with 15 units of Bst NI (10 units/. mu.l, New England Biolabs), 100. mu.g/ml bovine serum albumin (New England Biolabs) and 3.5. mu.l of 10 XNEB 2 buffer (New England Biolabs) in a total reaction volume of 35. mu.l. These reactions were covered with 20. mu.l of mineral oil and incubated overnight at 60 ℃. All reactions were analyzed by electrophoresis on a 5% Nusieve GTG gel (FMC Bioproducts, Rockland, Md.) and photographed with a Paoli camera.

In all reactions digested with Bst NI after PCR, it was seen that the 103bp fragment generated by amplification with primers 5BKIM and 3AKIL was only visible in the presence of 1: 10, 1: 10²And 1: 10³The ratio of Calu I to K562 DNA. The 103bp fragment could not be found to contain 1: 10⁴The reaction with Calu I: K562DNA at the ratio was not found in the reaction with only K562 DNA. The 73bp fragment resulting from digestion of the wild type amplicon by Bst NI was seen in all reactions. In the PCR after digestion with Bst NI reaction, 103bp fragment presence can diagnose K-ras codon 12 mutation presence.

When the ratio is 1: 10³The sensitivity of this method in the presence of the ratio of Calu I: K562DNA allows the detection of mutant Calu I DNA. Under these reaction conditions, inclusion of Bst NI in the PCR reaction resulted in preferential amplification (enrichment) of the mutant sequence but did not result in complete inhibition of amplification of the wild-type K562 sequence. Therefore the reaction was digested with Bst NI before best analysis. This method has moderate simplicity between the standard enrichment PCR method (requiring two rounds of PCR plus intermediate restriction endonuclease digestions to enrich for mutated sequences) and the standard REMS-PCR method (in which amplification of the wild-type sequence is completely inhibited and no subsequent manipulation such as digestion is required prior to analysis).

Discussion of the related Art

In the REMS-PCR method, the restriction endonuclease and the DNA polymerase must i) function in the same reaction conditions (e.g., salt, pH) that must be compatible with PCR and ii) be sufficiently thermostable in these reaction conditions to maintain activity during the thermal cycling required for PCR. Some of the restriction endonucleases listed in table 1, as well as other heat-resistant restriction endonucleases, would be suitable for incorporation into the REMS-PCR method if buffer conditions were identified that i) are compatible with the restriction endonuclease activity and maintain endonuclease activity when the reaction is thermal cycling during PCR and ii) are compatible with simultaneous DNA polymerase activity and maintain polymerase activity when thermal cycling during PCR.

Since little is known about the ability of restriction endonucleases to maintain activity during the thermal cycling required for PCR, a simple and easy analytical method was developed to identify candidate thermostable restriction endonucleases and reaction conditions. In the activity/thermostability assay, the enzyme activities of restriction endonucleases in various reaction conditions can be compared after a certain number of thermal cycles. In this assay, a reaction solution containing a primer of standard PCR concentration, dNTP, and DNA polymerase is prepared. The reaction solution does not contain template DNA but includes a buffer system, with or without additional reagents, and the restriction endonuclease to be detected. The reaction was placed in a thermocycler, raised to high temperature and then thermocycled, the reaction was terminated after a certain number of thermocycles, plasmid DNA was added to the tube and the reaction was incubated at the optimum temperature for the restriction endonuclease specified by the manufacturer. The enzymatic activity of the restriction endonuclease can be assessed by observing the degree of cleavage of the plasmid DNA on gel electrophoresis.

The activity/thermostability assay identified various restriction endonucleases, including Bst NI, BslI, Tru91, and Tsp509I, that were sufficiently thermostable in some buffer conditions to maintain moderate or complete catalytic activity after the thermal cycling necessary for PCR. The most effective reaction conditions for maintaining catalytic activity during thermal cycling were identified. The catalytic activity of the restriction endonuclease after thermal cycling depends on the pH and ionic strength of the buffer, the monovalent cation (K)⁺Or N⁺) Selection and concentration of free Mg²⁺And the presence of other additives including Dithiothreitol (DTT). The effect of each of these components is dependent on the other components in the buffer.

It is also likely that the enzyme activity of the restriction endonuclease is maintained by reducing the temperature and time for DNA denaturation during PCR. Factors known to affect the melting temperature of duplex DNA molecules include salt concentrations and the presence of reagents such as formamide, dimethyl sulfoxide, glycerol and ethylene glycol, which are compatible with at least some PCR systems. Inclusion of these or other reagents that affect the melting temperature of DNA may allow PCR to be completed at reduced denaturation temperatures and/or times. These agents may also have a direct positive or negative effect on the activity and/or thermostability of the restriction endonuclease (and/or DNA polymerase). The effect of various thermal cycling temperature profiles on restriction endonuclease activity in the presence of additional reagents can be assessed by the thermotolerance/activity assay described above. Additional thermostable restriction endonucleases, and identification of reaction conditions that maintain restriction endonuclease activity during thermocycling can be accomplished using conventional activity/thermostability assays without the need for creative efforts.

For REMS-PCR, the reaction conditions must not only maintain the catalytic activity of the restriction endonuclease but they must be suitable for PCR, and thus the buffer conditions must be compatible with the activity and heat resistance of the DNA polymerase during thermal cycling. There are many commercially available DNA polymerases available for PCR, and the general properties of these enzymes vary widely, including their optimal buffer conditions and the range of conditions they can tolerate. Detection of the efficiency of various DNA polymerases in PCR under reaction conditions known to maintain restriction endonuclease activity allows for the identification of compatible DNA polymerase/restriction endonuclease/buffer combinations. It has been demonstrated that the range of reaction conditions that maintain the restriction endonuclease activity also analyzed their compatibility with PCR using various primers and various DNA polymerases. The effect of different components of the reaction conditions on the PCR varies from primer pair to primer pair and may depend on other reaction components. For this purpose, the specific primer set required for PCR should be detected in this manner. PCR conditions compatible with the simultaneous activities of both the restriction endonuclease and the DNA polymerase and resulting in efficient amplification with a particular primer pair can be identified by routine and non-inventively skilled testing.

REMS-PCR requires a heat-resistant restriction endonuclease recognition/cleavage site spanning the nucleotide of the genetic variation to be analyzed, which may either occur naturally or be induced by primers containing internal mismatches to the template. When the restriction endonuclease recognition/cleavage site is induced by a primer, the site is located partially within the primer and partially within the synthetic sequence 3' of the primer in the amplicon. Thus, the primer must include any mismatched base that is required to induce a restriction nuclease site, but should not overlap with the base to be analyzed. Rules for designing PCR primers containing mismatched bases near the 3' end have been established (S.Kwok, et al 1990, nucleic acids Res. 18, 999-. Although some end-mismatched primers will not amplify efficiently and reduce the yield of specific amplicons by 100-fold, most will amplify as efficiently as fully paired primers. For example, when the terminal 3' base in the primer is G, it extends over a template containing C, T or G at the complementary position, but not over a template containing A at the complementary position.

Recognition/cleavage sites can be more easily induced when restriction endonucleases require only a short four-nucleotide recognition sequence (e.g., Tru9I or Tsp509I) or when they recognize multiple sequences (e.g., Bst NI). Recognition/cleavage sites for restriction endonucleases recognizing interrupted short sequences are particularly susceptible to induction, e.g., the Bsl I recognition sequence CCNNNNNGG, where N is any nucleotide. Bsl I can be used to analyze mutations that occur at codons encoding glycine (GGN) or proline (CCN). In general, primers designed to induce a BslI recognition site in these codons can be extended by DNA polymerase because they will not require mismatched bases near the 3' end and single or double mismatched bases located in the middle of the primer sequence will tolerate and often not inhibit PCR amplification.

In addition, one skilled in the art can design primers that induce BslI recognition sites for analysis of most (about 80%) of the mutations that occur in G or C. Mutations at bases G and C are common, for example, the percentage of p53 mutations that occur at the G or C base is at least 77% in colon cancer mutations, at least 72% in lung cancer mutations, at least 74% in bladder cancer mutations, at least 61% in breast cancer mutations and at least 66% in brain tumor mutations (M.Hollstein et al 1996 nucleic acid Res. 24, 141-146). The following table lists all possible combinations of sequences around bases C or G and the terminal bases required for primers to induce CC or GG as part of the Bsl I site at these positions. Template/primer combinations expected to be compatible with PCR are shown in Table 15.

Watch 15

The template sequence adjacent to the target base (underlined) is N ═ a, C, G, TX ═ a, C, TY ═ a, G, T	Primer type sense (5 'primer) antisense (3' primer)	Primer 3' base	Template 3' base	Compatibility with PCR
					GGN	Sense of	G	C	Is that
AGN	Sense of	G	T	Is that
					CGN	Sense of	G	G	Is that
TGX	Sense of	G	A	Whether or not
					NGG	Sense of	N is the same as each template (sense)	N-as per template (antisense)	Is that
NCC	Antisense gene	G	C	Is that
					NCT	Antisense gene	G	T	Is that
NCG	Antisense gene	G	G	Is that
					YCA	Antisense gene	G	A	Whether or not
CCN	Antisense gene	N-as per template (antisense)	N is the same as each template (sense)	Is that

Examples of recognition/cleavage sites for natural or induced heat-resistant restriction endonucleases in genes associated with acquired diseases are listed in Table 16, and in these examples, restriction endonucleases recognizing wild-type sequences were identified. The table includes restriction endonucleases known to be compatible with REMS-PCR and other endonucleases that may be compatible with the present method. The primers used for these mutation analyses must include the base to be induced (in bold) but not overlap with the base to be analyzed (underlined). Ras-proto-oncogenes (K-Ras, H-Ras and N-Ras) are frequently activated in many human cancers by acquiring point mutations at codons 12, 13 and 61. Since codons 12 and 13 of all three ras genes encode glycine, Bsl I can be used to analyze most ras-mutations. A novel point mutation in intron D of H-ras has also been found in bladder cancer. Resistance of HIV strains to some drugs is associated with acquisition of point mutations.

TABLE 16

Gene	Disease and disorder	Reason	Wild type sequence (base to be analyzed) restriction endonuclease recognition site and name (base to be induced)
				K-rasN-rasH-ras	Cancer treatment	Point mutations at codons 12 and 13, e.g., k-ras codon 12, e.g., k-ras codon 13	GTTGGAGCTGGCCNNNNNNGG Bsl IGGAGTCCTGGCCNNNNNNNGG Bsl I
K-ras	Cancer treatment	Point mutation of codon 12	GCTGGCCTGG BstNI
				K-rasN-rasH-ras	Cancer treatment	Codon 61-position 1 point mutations such as H-ras	CCAGGAGGAGTCCNNNNNNNGG Bsl 1
H-ras	Cancer treatment	Codon 61-position 3 Point mutation	GGCCGGCCAGGCCNNNNNNNGG Bsl 1
				H-ras	Cancer treatment	Point mutation of codon 61 (except A to T at position 2)	CCAGGCCAGG(CCTGG) BstN1
H-ras	Cancer of the bladder	Point mutation of intron D	GTAATTAA Tru 91
				HIV-I	Resistance to AZT	Point mutation 1. codon 412. codon 703. codon 215	1.GAAATGAATT Tsp 509IGCAATG Bsr DI2.AAATGGAATT Tsp 509I3.TTTACCTTAA Tru 9I
	ddI resistance	Point mutation of codon 74	AAAATTAAATT Tsp 509I

The selection of a set of genes that may carry heritable mutations associated with a disease is listed in table 17. The listed sequences are either wild type or mutant and the possible positions of sequence variation are underlined. The analysis of recessive mutations requires the differentiation of heterozygous carriers from homozygous individuals, which are at risk for disease development. For all the following examples, restriction endonucleases recognizing the wild type sequence were identified. For cystic fibrosis transductant genes, restriction endonucleases that recognize the mutated sequence have also been identified. TABLE 17

Gene	Disease and disorder	The sequence to be analyzed	Sequence type/sequence (base to be analyzed) endonuclease site and name (base to be induced)
				Cystic fibrosis transmembrane conductance regulator	Cystic fibrosis	The point mutation is located at 1, codon 5422, codon 5513, IVS-44, deletion codon 508(3bp)	The wild type sequence 1.ATAGTTCTTGGCCNNNNNNNGG Bsl ICCTGG BstNI2. CTGAGTGGAGGTCACCNNNNNNNNGG Bsl IGGTCC BsiZI3. TTATAAGAGGCCNNNNNGG Bsl I4. AAATATCTTGATNNNATNNATC Bsa BIBsiBI
Wild type sequence 1, codon 5422, codon 5513, IVS-44, codon 508	Mutant sequence 1, TCTTTGATTAA Tru9I 2 GATCAACGAGGATGNANNNNATC Bsa BIBsi BI3 AAGAAGTTAATTAA Tru9I 4 AAATATCATGTGGCCNNNNNNNNNNGG Bsl I
		Alpha-trypsin	Hepatofibrosis emphysema			Codon 342 Point mutation	Wild type sequence GACCATCGACGCCNNNNNNNGG Bsl I
Beta-globulin	Beta-thalassemia			Point mutation IVS-1 (beta-Mediterranean)	Wild type sequence CCCTGGGCAGGCCNNNNNNNGG Bsl I
		Point mutation polyA signal (. beta. + -Black)	Wild type sequence AATAAATTAA Tru9I

Little is previously known about the role of including heat-resistant restriction endonucleases in PCR. It has been found that having both restriction endonuclease and DNA polymerase activities in the PCR process results in (i) inhibition of amplification of sequences containing restriction endonuclease recognition/cleavage sites and (ii) selective amplification of variants of such sequences lacking restriction endonuclease recognition/cleavage sites, which has led to the development of a method known as REMS-PCR which can be used to analyze acquired or genetic polymorphisms, including point mutations, small deletions and insertions. When the REMS-PCR method is designed to detect mutant sequences, the wild type, but not the mutant subsequence contains the recognition/cleavage sequence of a heat-resistant restriction endonuclease. PCR amplification of the wild-type sequence is inhibited by the activity of the restriction endonuclease. In contrast, the mutant sequences are selectively amplified by DNA polymerase during PCR.

The REMS-PCR method can also be designed to selectively inhibit the amplification of the mutant rather than the wild-type sequence. If the REMS-PCR method is designed to detect wild-type sequences, the mutant, but not the wild-type sequence, contains a heat-resistant restriction endonuclease recognition/cleavage sequence. PCR amplification of the mutant sequences will be inhibited by the activity of the restriction endonuclease and the wild type sequences will be selectively amplified by PCR. Failure to amplify a specific wild-type sequence indicates a homozygous mutation. Detection of both wild type and mutant sequences indicates the presence of heterozygous mutations.

Several REMS-PCR methods were developed for the analysis of point mutations at codon 12 of the K-ras oncogene. These methods utilize both Bst NI and DNA Taq polymerase, or Bst NI and Stoffel fragment polymerase, or BslI and DNA Taq polymerase, which are enzymatically active. These methods include multiplex primer systems comprising diagnostic primers and one or two sets of control primers. If codons 12, positions 1 and 2 are wild-type, the diagnostic primers induce a recognition/cleavage site for BstNI or BslI in the K-ras amplicon. The inclusion of one of these restriction endonucleases in the PCR results in suppression of amplification of the wild type DNA template and selective amplification of DNA templates containing mutations at positions 1 or 2 of codon 12, so that amplification with these primers can diagnose for the presence of a codon 12 point mutation. Additional control primers were included in all reactions to confirm the reaction conditions, including the amount of template DNA sufficient for amplification by PCR. These PCR control primers can flank any region that does not contain a restriction endonuclease recognition/cleavage site, and the amplicon that incorporates these primers must be present for a clear interpretation of the negative results. A second control primer was included in a multiplex system to confirm that the restriction endonuclease mediated complete inhibition of PCR amplification. The control primer for the restriction endonuclease must either induce or flank the recognition/cleavage site of the restriction endonuclease used in the REMS-PCR method. The absence of amplicons incorporating these primers clearly indicates a positive result.

The limit of the REMS-PCR assay was assessed by analyzing a sample containing CaluI DNA (heterozygous mutant for K-ras codon 12) diluted in Sup T1DNA (K-ras codon 12 wild-type) in the presence of Bst NI and DNA Taq polymerase. Detection of the diagnostic amplicon indicates the presence of the K-ras sequence at codon 12. Diagnostic amplicons were visualized by gel electrophoresis and colorimetric analysis in samples containing 1: 10 to 1: 10,000 ratio of CaluI: Sup T1, rather than Sup T1 alone. PCR control amplicons were detected on all samples including Sup T1 DNA. The literature indicates that this level of sensitivity will be sufficient for analysis of DNA extracted from clinical specimens including tissue resection and biopsy material, cytological samples and body fluids/secretions such as stool, urine and sputum containing small amounts of de-epithelialized tumor cells (D.Sidranky et al, 1992 science 256, 102-1; L.Mao et al, 1994 cancer research, 54, 1634-. Analysis of clinical specimens using REMS-PCR has been demonstrated. Mutations at K-ras codon 12 were detected in 2 of 4 colon adenocarcinomas but not in 4 normal colonic mucosa.

In the extension of REMS-PCR, this method can be accomplished using primers that simultaneously induce i) restriction endonuclease recognition/cleavage sites that are present only in the wild-type sequence and ii) multiple recognition/cleavage sites for restriction endonucleases specific for all possible mutated sequences. Subsequent partitioning of the diagnostic amplicons with restriction endonucleases confirmed the presence of mutations in these amplicons and the identification of the exact nucleotide substitutions in all cases.

It is also possible to develop such a REMS-PCR system: which results in selective amplification of the mutant sequence but does not result in inhibition of amplification of the wild-type sequence or vice versa. Thus, the reaction needs to be digested with the appropriate restriction endonuclease after PCR prior to analysis. This method has moderate simplicity between the standard enrichment PCR method and the standard REMS-PCR method where amplification of wild type sequences is completely inhibited.

REMS-PCR is compatible with a variety of capture and detection systems, which enable automation of the overall process and thus rapid analysis of large numbers of samples. Examples of capture systems include, but are not limited to i) PCR primers with GCN4 signature captured on GCN4 coated plates; ii) capturing the biotin-labeled primer with avidin or streptavidin; iii) digoxin-labeled product captured with anti-digoxin antibody; and iv) complementary oligonucleotides attached to latex beads or magnetic beads. Examples of detection systems include, but are not limited to, i) biotin-labeled PCR primers viewed with streptavidin/horseradish peroxidase; ii) direct labeling with fluorescein isothiocyanate or alkaline phosphatase molecules; and iii) a digoxin-labeled product detected with an anti-digoxin antibody.

REMS-PCR provides a sensitive and rapid method suitable for analyzing genetic variations associated with disease. The ability to simultaneously maintain restriction endonuclease and DNA polymerase activities during PCR allows the development of a simple method for selectively amplifying variant sequences in reactions that contain all reagents, including all enzymes, at the start of PCR. The reaction can be done in a closed system to reduce the chance of contamination during the PCR reduction process. The EMS-PCR method has fewer steps than other methods using restriction endonuclease mediated selective amplification and/or analysis of mutated sequences. Generally, the reaction requires no further manipulation prior to detection, however, the method does not preclude subsequent analysis of the diagnostic amplicon in order to identify the exact nucleotide substitution. The selective amplification and reduction in the number of steps required for analysis with restriction endonucleases makes the REMS-PCR analysis fast, labor-saving and more amenable to automation.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Reference to the literature

The Ki-ras 2 gene is activated by 2 different point mutations in human colon and lung cancer by Capon, d.j., Seeburg, p.h., Mc Garth, j.p., Hayflick, j.s., Edmun, u. 304, 507 and 513.

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Levi, S., Urbano-Ispizua, A., Gill, R., Thomas D.M., Gilbertson J., Foster C and Marshall C.J. (1991), multiple K-ras codon 12 mutations in cholangiocarcinoma demonstrated with sensitive polymerase chain reaction techniques. Cancer study, 51, 3497-.

Mao, L., Hruban R.H., Boyle J.O., Tockman M., and Sidransky D. (1994) detection of mutations in oncogenes in sputum preceded the diagnosis of lung cancer. Cancer research, 54, 1634-.

Saiki, r.k., Scharf, s., F aloona, f., Mullis, k.b., Horn, g.t., Erlich, h.a., and ameim, N. (1985) restriction site analysis of beta-globin genomic sequences and for the diagnosis of sickle cell anemia. Science 230, 1350-.

Sambrook, j., Fritsch, e.f., and manitis, T. (1989) molecular cloning experimental manual, second edition, new york: cold spring harbor laboratory Press.

Antibodies as thermolabile switches by Sharkey, d.j., scale, e.r., Christy, k.g.jr., Atwood, s.m., and Daiss, j.l. (1994): the polymerase chain reaction is initiated at high temperature. Bio/technology 12, 506-.

Sidransky, D.D., Tokino T.S.R., Hamilton S.R., Kinzler K.W., Levin B.Frost P.and Vogelstein B.1992) cured stool from colon cancer patients for the identification of ras oncogene mutations. Science 256, 102-.

Singh, j., Rao, c.v., Kulkarni n., Simi b., and Reddy B.S, (1994) as molecular markers for transition endpoints in colon cancer chemoprevention: regulation of ras activation is caused by sulindac and phenylhexane iso-sulfatide during colon carcinogenesis. International journal of cancer 5, 1009-.

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Claims (12)

1.A method of detecting a genetic polymorphism in an individual, the method comprising the steps of:

(1) obtaining a sample containing individual nucleic acids;

(2) amplifying the nucleic acid sample of step (1) by a method comprising thermal cycling and primers, the amplification being carried out in the presence of a thermostable restriction endonuclease, which enzyme retains activity during thermal cycling, the primers being selected such that they introduce into nucleic acid amplified from nucleic acid not comprising the polymorphism or from nucleic acid comprising the polymorphism a sequence recognized by the thermostable restriction endonuclease; and

(3) analyzing the product of step (2) to detect the presence or absence of the polymorphism.

2. The method of claim 1, wherein the primer introduces a sequence recognized by a heat-resistant restriction endonuclease into a nucleic acid amplified from a nucleic acid that does not include the polymorphic nucleic acid.

3.A method for detecting a genetic polymorphism in an individual, the method comprising the steps of:

(1) obtaining a sample containing individual nucleic acids;

(2) amplifying the nucleic acid sample of step (1) by a method comprising thermal cycling and primers, the amplification being carried out in the presence of a heat-resistant restriction endonuclease which is active at the same time, the restriction endonuclease being selected such that it recognizes nucleic acid which does not comprise a polymorphism but does not recognize nucleic acid which comprises a polymorphism or such that it recognizes nucleic acid which comprises a polymorphism but does not recognize nucleic acid which does not comprise a polymorphism; and

(3) analyzing the product of step (2) to detect the presence or absence of the polymorphism.

4. The method of claim 3, wherein the thermostable restriction endonuclease recognizes a nucleic acid that does not include a polymorphism.

5. The method of claim 2 or claim 4, wherein the method further comprises the additional step of:

(4) reacting the amplified nucleic acid of step (2) with at least one restriction endonuclease selected to digest amplified nucleic acid comprising the particular polymorphism; and

(5) detecting whether digestion has occurred in step (4), digestion indicating the presence of a particular polymorphism.

6. A method according to any one of claims 1 to 5 wherein the method comprising thermocycling is PCR.

7. The method according to any one of claims 1-6, wherein the analysis of step (3) comprises detecting the presence or absence of the amplified nucleic acid of step (2), the presence or absence of the amplified nucleic acid indicating the presence or absence of the polymorphism.

8. A method according to any one of claims 1 to 7 wherein the nucleic acid is DNA.

9. The method of any one of claims 1-8 wherein the thermostable restriction endonuclease is selected from the group consisting of Bst NI, BslI, Tru9I, and Tsp 509I.

10. The method of any one of claims 1-9, wherein the genetic polymorphism is detected in one of the ras proto-oncogenes K-ras, N-ras and H-ras, or p53 tumor suppressor gene.

11. The method of claim 10 wherein the genetic polymorphism is detected in codon 12 of K-ras.

12. A method according to any one of claims 1 to 9 wherein the genetic polymorphism is detected in HIV-I, cystic fibrosis transmembrane conductance regulator, alpha-antitrypsin or beta-globin.