Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-13
2001-03-06
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06197509
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns analysis of DNA particularly for examining genetic markers, which is useful in, for example, forensic medicine, population studies, family linkage studies and disease diagnosis.
BACKGROUND TO THE INVENTION
It is known that there are simple nucleotide sequences in the human genome that can occur in different numbers of repeats in different individuals, giving rise to a range of different alleles or variants of different length that can be used as genetic markers to typify the DNA of an individual.
Tandem repeat minisatellite and microsatellite regions in vertebrate DNA frequently show high levels of allelic variability in the number of repeat units. These highly informative genetic markers have found widespread applications in population genetics, forensic science, medicine and other natural scientific studies. For example, these markers can be used for linkage analysis, determination of kinship in paternity and immigration disputes and for individual identification in forensic medicine. In a minisatellite system, a core DNA sequence unit is usually 15 or more base pairs. To date most studies and applications of such systems have relied on Southern blot estimation of allele length, which requires at least 50 ng of relatively undegraded DNA. It is often very difficult to extract such large amounts of DNA from many forensic samples such as blood and semen stains.
Microsatellites, on the other hand, are short tandemly repeated (STR) polymorphic DNA sequences which are most commonly in the form of dinucleotide repeats such as (dC-dA)n, but can also be trinucleotide and tetranucleotide repeats. For a further discussion, see Pena. S. D. J. and Chakraborry, R. (1994). Paternity testing in DNA era. Trends in Genetics Vol.10, 204-209. Microsatellites can be amplified using the polymerase chain reaction (PCR) and the resulting ampileons normally range from 80-800 base pairs (bps) in length and so are well suited to processing in automated sequencing machines which are now widely used for gene scanning and typing. (See Read, P. W. et al (1994), Chromosome-specific microsatellite sets for fluorescence based, semiautomatic genome mapping. Nature Genet. 7,390-395.) To date, most microsatellite polymorphisms have been based upon dinucleotide repeats. Because of the very small size difference between adjacent alleles, some of the results can be difficult to interpret. Tri and tetranucleotide repeats are easier to use but occur less frequently in the human genome. Expansion of trinucleotide repeat sequences has also been implicated in a number of genetic diseases, including Huntingdon's disease, fragile X syndrome and myotoaic dystrophy.
The present invention is based on the discovery in the human inducible nitric oxide synthase (iNOS) gene of a pentanucleotide repeat (CCTTT/GGAAA)n. The repeat is located approximately 2.8 kb 5′ end of upstream promotor region of the iNOS gene on 17q11.1-q11.2. Investigations have shown this pentanucleotide repeat (which is referred to for convenience as Xu-1) occurs in widely varying numbers in different individuals; so far, 12 different variants or alleles have been detected, having between 7 and 18 contiguous Xu-1 repeats. The different alleles are referred to as A
7
, A
8
. . . A
18
. Because the Xu-1 repeat is highly polymorphic in the human population, the repeat leads itself to use as a microsatellite marker with uses in, for example, forensic medicine, population studies, family linkage studies and disease diagnosis.
SUMMARY OF THE INVENTION
In one aspect the present invention provides an a method of analysing a sample of DNA to determine the number of contiguous repeats of the sequence (CCTTT/GGAAA) in the iNOS gene.
The number of repeats is typically in the range 7 to 18.
By analysing DNA in this way, a determination can be made of the number of Xu-1 repeats in a particular DNA sample, that is which allele or alleles (usually one or more of A
7
to A
18
) are present
The sample may be, for example, a sample of blood, semen, saliva, buccal cells or any other suitable biological material.
Because the Xu-1 repeat is a pentanucleotide repeat, it is easier to distinguish adjacent alleles simply on the basis of size than is the case for smaller repeating units. Experiments have also shown that there is considerable variation in the two alleles (one from each chromosome) of different individuals. The heterozygosity has been calculated as 0.841. The Xu-1 repeat is therefore highly polymorphic and hence has significant valve as a genetic marker (the Xu-1 marker) that is easy to use.
Samples are conveniently analysed by use of the polymerase chain reaction (PCR), enabling the method of the invention to be performed on small quantities of sample. A pair of PCR primers has been designed for this purpose, generating products in the range about 170 to 225 base pairs (bp) in size. The forward primer is 5′-ACCCCTGGAAGCCTACAACTGCAT-3′ (Seq. ID No. 1). The reverse primer is 5′-GCCACTGCACCCTAGCCTGTCTCA-3′ (Seq. ID No. 2).
The resulting products may be sequenced to determine the number of Xu-1 repeats. Alternatively, fragment length can simply be determined, eg by running on an electropheretic gel, enabling calculation of the number of Xu-1 repeats.
Heterozygosity can be increased substantially by using the Xu-1 marker in conjunction with another genetic marker. For example, good results have been obtained using the Xu-1 marker with a known microsatellite marker based on the polymorphic trinucleotide repeat (ATT/TAA) present in the neuronal nitric oxide synthase (NOS1) gene in repeat numbers ranging from 5 to 13. PCR primers have been designed for use with the NOS1 marker to generate products in the range 110 to 138 bp (ie distinct in size from the Xu-1 marker products), that can be used under the same PCR conditions as the Xu-1 primers. The forward primer is 5′-GAAATTGGTCATAGTGGGAATG-3′ (Seq. ID No. 3). The reverse primer is 5′-GTGTTGGTGAACCAACCCTCCTAA-3′ (Seq. ID No. 4).
PCR reactions for the 2 markers can thus be run together, and by using different labels (eg green and blue) for the primers, the PCR products can be detected simultaneously on the same gel. By using these 2 markers together, heterozygosity is incresed to about 99%.
Experiments have shown that there are significant differences in the distribution of alleles with different numbers of the Xu-1 repeat in different ethnic groups. The Xu-1 marker may therefore be of value in population studies, immigration disputes, paternity determination and forensic studies.
Furthermore, because the Xu-1 marker is located in the 5′ end of human iNOS gene, which has been implicated in certain common human diseases, such as Alzheimer's disease, hypertension, diabetes and cancers, the marker can be used in allelic association studies and mutation analysis for the diseases. For example, a strong allelic association has already been detected between the repeat number and the senile dementia Lewy body (SDLT) variant of Alzheimer's disease, which represents about one quarter of all cases of Alzheimer's disease. Mutations in flanking sequences have also been detected in some cases of colon cancer. The iNOS gene is also involved in tissue transplantation. The polymorphic marker described in this invention also could be used in genotype typing for tissue transplantation.
The invention will be further described, by way of illustration, in the following Examples and by reference to the accompanying Figures.
REFERENCES:
patent: 5882908 (1999-03-01), Billiar et al.
Spitsin, S.V. et al. Molecular Medicine 2(2):226-235, Mar. 1996.
Spitsin, S.V. et al. GenBank Accession No. Z49251, Jan. 1997.
Johannsen Diana
Medical Research Council
Myers Carla J.
Pillsbury Madison & Sutro
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