Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-16
2001-07-17
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091100, C435S091500
Reexamination Certificate
active
06261768
ABSTRACT:
FIELD OF INVENTION
This invention relates to methods for in vitro amplification of specific nucleic acid target sequences. In particular the invention relates to methods which employ thermophilic restriction endonucleases to mediate selective amplification of nucleic acid targets which contain sequence differences including point mutations, deletions and insertions.
BACKGROUND OF THE INVENTION
A variety of inherited and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Some of these variants are directly associated with the presence of disease, while others correlate with disease risk and/or prognosis. There are of the order 500 human genetic diseases which result from mutations in single genes. These include cystic fibrosis, muscular dystrophy, &agr;1-antitrypsin deficiency, phenylketonuria, sickle cell anaemia or trait, and various other haemoglobinopathies. Furthermore, individuals with increase susceptibility to several common polygenic conditions, such as atherosclerotic heart disease, have been shown to have an association with the inheritance of a particular DNA sequence polymorphism. Cancer is thought to develop due to accumulation of lesions in genes involved in cellular proliferation or differentiation. The ras proto-oncogenes, K-ras, N-ras, and H-ras, and the p53 tumour suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes leads to activation or increased transforming potential. Genetic analysis is likely to become routine in the clinic for assessing disease risk, diagnosis of disease, predicting a patient's prognosis or response to therapy, and for monitoring a patient's progress. The introduction of such genetic tests depends on the development of simple, inexpensive, and rapid assays for genetic variations.
In rare instances mutations can be detected if they happen to lie within a naturally occurring restriction endonuclease recognition/cleavage site. WO84/01389 describes a method for discriminating between wild type genes and non wild type variants by screening for the presence or absence of restriction endonuclease sites. The inventors demonstrated the principle by analysis of variant sequences at condon 12 of the human H-ras proto-oncogene. The wild sequence at condon 12 forms part of the recognition/cleavage sites for the restriction endonucleases Nae I and Hpa II. Digestion with these endonucleases can discriminate between the wild type proto-oncogene and activated oncogenes which harbour mutations at this condon. Point mutations at condon 12 of H-ras are frequently found in bladder carcinomas and this general strategy form the basis of screening kits for medical diagnosis.
Methods of in vitro nucleic acid amplification have wide-spread applications in genetics and disease diagnosis. The polymerase chain reaction (PCR) is a powerful, exquisitely sensitive procedure for in vitro amplification of specific segments of nucleic acids (R. K. Saiki, et al 1985 Science 230, 1350-1354 of F. F. Chehab, et al 1987 Nature 329, 293-294 and U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195 and U.S. Pat No. 4,800,159 and U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,176,995). The PCR is mediated by oligonucleotide primers that flank the target sequence to be synthesized, and which are complementary to sequences that lie on opposite strands of the template DNA. The steps in the reaction occur as a result of temperature cycling (thermocycling). Template DNA is first denatured by heating, the reaction is then cooled to allow the primers to anneal to the target sequence, and then the primers are extended by DNA polymerase. The cycle of denaturation, annealing and DNA synthesis is repeated many times and the products of each round of amplification serve as templates for subsequent rounds. This process results in the exponential amplification of amplicons which incorporate the oligonucleotide primers at their 5′ termini and which contain newly synthesized copies of the sequences located between the primers.
The PCR is extremely versatile and many modifications of the basic protocols have been developed. Primers used for PCR may be perfectly matched to the target sequence or they can contain mismatched and or modified bases. Additional sequences at the 5′ end or primers can facilitate capture of PCR amplicons and the inclusion of labelled primers can facilitate detection. The inclusion of mismatched bases within primers can result in the induction of new restriction endonuclease recognition/cleavage sites. These sites can be located completely within the primer sequence. Alternatively, they can span a sequence which lies partially within the primer and partially within the newly synthesized target sequence (J. B. Cohen and A. D. Levinson (1988) Nature 334, 119-124). The general rules for designing primers which contain mismatched bases located near the 3′ termini have been established (S. Kwok, et al. (1990) Nucleic Acids Research 18, 999-10005).
Modified primers containing mismatched bases were used to induce novel recognition/cleavage sites for restriction endonucleases in H-ras amplicons which were mutated at codon 12 (R. Kumar and M. Barbacid (1988) oncogene 3, 647-651). Similarly, primers containing mismatched bases were employed in protocols known as allele specific enrichment (Todd A V et al Leukemia, 1991; 5:160) or enriched PCR (Levi S et al Cancer Res., 1991; 6:1079). These are very sensitive protocols for the detection of point mutations. In these protocols, DNA samples were amplified with primers which induced either an Eco NI site in N-ras amplicons, or a Bst NI site in K-ras amplicons, provided the sequences were wild type at condon 12. Aliquots of the PCR reactions were digested with the appropriate restriction endonuclease to cleave wild type amplicons prior to re-amplification of the digestion-resistant amplicons in a second round of the PCR. These protocols resulted in preferential amplification of sequences harbouring point mutations at condon 12 of ras. More recently, a simplified enriched PCR protocol was published which allowed the reaction to be performed in a single tube (Singh et al Int J Oncol., 1994; 5: 1009). This protocol also required an initial round of PCR amplification, however, the restriction endonuclease was then added directly to the reaction tube. Following incubation with the restriction endonuclease, a second round of the PCR resulted in amplification of sequences harbouring mutations within the restriction endonuclease recognition/cleavage site. This analysis of natural or induced restriction endonuclease sites in PCR amplicons requires sequential activity of a DNA polymerase for the PCR, followed by activity of a restriction endonuclease for cleavage analysis. Enriched PCR protocols require sequential activity of firstly a DNA polymerase for the PCR, then restriction endonuclease activity to cleave specific sequences, followed by further DNA polymerase activity to re-amplify digestion resistant amplicons.
The ability to simultaneously exploit the activities of a restriction endonuclease and a DNA polymerase during the PCR could provide several advantages. It could allow the development of simple protocols for exclusive or preferential amplification of variant sequences in reactions which contain all reagents, including enzymes, at the initiation of the PCR. It was not previously known whether or not inclusion of a restriction endonuclease in a PCR could result in (i) complete (or partial) inhibition of amplification of a sequence which contains the recognition/cleavage site for the restriction endonuclease and (ii) exclusive (or preferential) amplification of a variant of this sequence which lacks the recognition/cleavage site for the restriction endonuclease. The ability to completely inhibit amplification of a sequence and/or exclusively amplify a variant sequence could lead to the development of protocols which do not require further manipulation prior to analysis. A reduction in the nu
Fuery Caroline Jane
Todd Alison Velyian
Browdy and Neimark
Johnson & Johnson Research Pty. Limited
Myers Carla J.
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