Selective ligation and amplification method

Details Selective ligation and amplification method Selective ligation and amplification method

1999-12-17

2001-06-12

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S005000, C435S091100, C435S091200, C435S091500, C435S094000, C435S183000, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06245505

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for in vitro amplification of specific nucleic acid target sequences. In particular the invention relates to methods which initially depend on concurrent DNA polymerase and ligase activity to mediate amplification of nucleic acid targets. The method can be used to selectively amplify nucleic acid sequences which contain sequence variations such as 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 more than 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 increased 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 the accumulation of genetic lesions in genes involved in cellular proliferation or differentiation. The ras proto-oncogenes, K-ras, N-ras, and H-ras, and the p53 tumor suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes result in an increase in 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.
Due to increasing interest in the development of such tests a number references have been published in this area, these include, Abravaya, K., Carrino, J. J., Muldoon, S. and Lee, H. H. (1995) Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Research 23, 675-682; Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. 88, 189-193; Belgrader, P., Marino, M. M., Lubin, M. and Barany, F. (1996) A multiplex PCR-Ligase detection reaction assay for human identity testing. Genome Science and Technology 1, 77-87; and Eggerding, F. A. (1995) A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. PCR Methods and Applications 4, 337-345.
In U.S. Pat. No. 5,593,840 there is disclosed a method of detecting a particular nucleic acid sequence. The method disclosed in this patent is said to be based on the discovery that certain aspects of polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used in combination to detect and amplify a target nucleic acid sequence. This method involves the use of three primers, with the third primer being complementary to at least a portion of the 5′ end of the first primer. It is an essential feature of this method that the position of the third primer complementary to the base at the 5′ end of the first primer contains a modification such as to substantially avoid strand displacement by polymerase.
Further information regarding PCR and LCR can be found in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, 5,176,995, EP 0 320 308 and EP 0 439 182 and the disclosure of these references is included herein by reference.
SUMMARY OF THE INVENTION
The present inventors have developed a sensitive and selective procedure for amplification of specific target nucleic acid sequences. The method involves both LCR and PCR. As the method involves Selective Ligation and PCR it has been termed “SLAP”.
Accordingly, in a first aspect the present invention consists in a ethod for amplifying a specific target nucleic acid sequence, the method comprising:
(1) forming a reaction mixture comprising:
(i) the target sequence;
(ii) primers comprising a first primer at least a portion of which at the 3′ end thereof is substantially complementary to a first segment at a first end of the target sequence, a second primer at least a portion of which at the 5′ end thereof is substantially complementary to a second segment at a second end of the target sequence, the 5′ end of the second primer being adjacent the 3 end of the first primer. and a third primer, the third primer being substantially complementary to a segment of the second primer at the 3′ end thereof;
(iii) at least four different nucleotide bases;
(iv) thermostable polymerase and thermostable ligase;
and
(2) thermocycling the reaction mixture.
In a second aspect the present invention consists in a method for detecting a specific target nucleic acid sequence in a sample comprising nucleic acid, the method comprising:
(1) forming a reaction mixture comprising:
(i) a sample of nucleic acid suspected to include the target sequence;
(ii) primers comprising a first primer at least a portion of which at the 3′ end thereof is substantially complementary to a first segment at a first end of the target sequence, a second primer at least a portion of which at the 5′ end thereof is substantially complementary to a second segment at a second end of the target sequence, the 5′ end of the second primer being adjacent the 3′ end of the first primer, and a third primer, the third primer being substantially complementary to a segment of the second primer at the 3′ end thereof;
(iii) at least four different nucleotide bases;
(iv) thermostable polymerase and thermostable ligase;
(2) thermocycling the reaction mixture; and
(3) detecting the presence or absence of an amplification product indicative of the presence of the specific target nucleic acid sequence.
In a preferred embodiment of the present invention the method further comprises. following the thermocycling of the reaction mixture the steps of substantially inactivating the thermostable polymerase and a subsequent second thermocycling of the reaction mixture.
In a preferred embodiment of the present invention the segment of the second primer to which the third primer is substantially complementary is a random sequence.
In a further preferred embodiment the reaction mixture includes a fourth primer which has the same sequence as a segment of the first primer at the 5′ end thereof.
The methods of the present invention can be used to simply amplify selected sequences. The method can also be used to determine the presence of particular sequences including for example to determine the presence of a mutation, allele, presence of a particular organism or virus. The target sequence can accordingly be from any source, including for example human and nonhuman animals, bacteria, yeast, fungi and viruses.
As used herein the term “random sequence” is intend to mean a sequence which is unrelated to the target sequence.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
DETAILED DESCRIPTION OF THE INVENTION (Strategy for Selective Ligation and PCR (SLAP))
In order that the nature of the present invention may be more clearly understood a preferred form thereof will now be described with reference to the following non-limiting general description of the operation of the method of the present invention and the following Example.
Figure Legends
FIG. 1
SLAP—Arrangement of primers in a 3 primer system.
FIG. 2
Location of the variant base in primers for SLAP.
FIG. 3
SLAP
FIG. 4
Selective ligation and PCR
FIG. 5
SLAP—arrangement of primers in a

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