Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-27
2002-11-12
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06479242
ABSTRACT:
BACKGROUND OF THE INVENTION
The most common polymorphisms in the human genome are single point mutations or single nucleotide polymorphisms also referred to as “SNPs”. An single nucleotide polymorphisms is a change, such as a deletion, insertion or substitution, in any single nucleotide base in the region of the genome of interest. Genotyping of single nucleotide polymorphisms will make possible genome-wide association studies, which are a powerful methods for identifying genes that make a contribution to disease risk.
For example, a point mutation G
1691
→A in the coagulation factor V gene, results in a Arg
506
→Gln amino acid mutation in the factor V molecule. This mutation, defined as factor V
LEIDEN
, leads to activated protein C (APC)-resistance and is the most common genetic risk factor for hereditary venous thrombosis.
Genotyping of single point mutations can be performed using a variety of assays, for example, a plasma based assay, Taqman, restriction digestion of PCR products, calorimetric mini-sequencing assay, radioactive labeled based solid-phase mini sequencing technique, allele-specific oligonucleotide (ASO), and single strand conformation polymorphism (SSCP). However, these methods are extremely time consuming.
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been employed to genotype single nucleotide polymorphism, in two assays, the Pinpoint assay and the Probe assay. In the PINPOINT assay a primer is annealed to the target DNA immediately upstream of the single nucleotide polymorphism site. The primer is extended in the presence of all four ddNTPs, but not dNTP. As a result, the primer is extended by a single base. The base at the single nucleotide polymorphism site is identified by the mass added onto the primer. However, this method suffers from poor resolution, as the mass difference between difference between the ddNTP nucleotides is quite small. For example, the mass difference between ddT and ddA nucleotides can be as little as 9 Da, rendering it difficult to identify the added nucleotide. Also, the presence of salt adducts of Na, Mg and K from the buffers employed in the assay renders it difficult to accurately measure the mass peak positions. Removing the salt adducts requires extensive time consuming desalting procedures.
Attempts have been made to address the resolution problems by using mass-tagged dideoxynucleotides in place of the unmodified dideoxynucleotides. However, the presence of such mass tags slows sequencing reactions.
Another attempt was made to overcome the resolution problem by adding a cleavable site to the primer that can be cleaved after extension to produce lower mass markers that can be identified in a more accurate manner. However, this still required extensive de-salting and added an additional step of cleaving the primer.
PROBE is a method in which the primer is extended in the presence of three deoxy nucleotides (dNTPs) and a one dideoxynucleotide which is complementary to the nucleotides of one of the alleles. In this way, the primer is extended by one base by the addition of the ddNTP from one allele. Primers annealed to target DNA containing the other allele are extended by the addition of dNTPs until the ddNTP is eventually incorporated and the primer is terminated. The number of dNTPs added depends on the nucleotide sequence of the target DNA. However, the probe assay suffers from low detection sensitivity and peak overlapping because of the long extension products.
It would be desirable to have an assay for determining single nucleotide polymorphisms which has high resolution, high detection sensitivity, does not suffer from long extensions, and does not require extensive desalting.
SUMMARY OF THE INVENTION
A new assay for genotyping of single nucleotide polymorphisms has been developed, which has the advantage of high resolution, high detection sensitivity, no need for labeling, and does not require extensive desalting steps. Such new method, hereinafter referred to as the “VSET assay”, tends to produce very short extension products. The VSET assay is accurate, fast, efficient and allows for simultaneous multiplex genotyping of a number of mutation sites. The VSET is also compatible with automation.
The VSET method of genotyping a nucleotide polymorphism comprises the following steps: providing a polynucleotide acid sample comprising at least one target site, and a first region of nucleotides immediately adjacent to the target site; preferably genomic DNA; preferably amplifying the polynucleotide; then combining the polynucleotide sample with: three dideoxynucletides selected from the group of ddGTP, ddATP, ddCTP, and ddTTP; and one deoxynucleotide selected from the group consisting of dGTP, dATP, dCTP, and dTTP wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and a mini-sequencing primer complementary to the first region of nucleotides; extending the mini-sequencing primer with a dideoxynucletide or deoxynulceotide whose base is complementary to the base at the target site, to provide extension products; and then identifying the extension products. It is highly preferred that the extension products be identified by mass spectrometry, preferably Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
In the VSET assay, mini-sequencing is conducted in the presence of: a nucleic acid polymerase, where the sample is DNA, then the polymerase is DNA polymerase; three dideoxy nucleotides; and one deoxynucleotide. One of the three ddNTPs is complementary to one of the allelic variations at the single point mutation site while the deoxynucleotide is complementary to the other allelic variant at the single point mutation site. The mini-sequencing primer hybridizes to a polynucleotide sequence immediately next to a single point mutation site and is extended by the polymerases. Typically, the extension products will contain either one or two nucleotides in addition to the primer sequence used in the mini-sequencing step. The genotype at the variable site is determined on the basis of the number of nucleotides contained in the extension products. This assay is particularly useful to genotype the factor V mutation in the Factor V gene, specifically the G
1691
→A mutation of Factor V.
In the preferred form of the method to genotype the factor V mutation in the Factor V gene, a fragment of genomic DNA containing the 1691
th
base is preferably first amplified, using conventional techniques, preferably conventional polymerase chain reaction techniques also referred to herein as “PCR”. Next, mini-sequencing of the amplified polynucleotide is conducted in the presence of primers complementary to the nucleotides adjacent to the nucleotide polymorphism, and dGTP, ddATP, ddCTP, and ddTTP, to provide extension products. The extension products are analyzed preferably using mass spectrometry, preferably matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. The base at position 1691 is identified based on the number of nucleotides added to the primer used in the mini-sequencing. Alternatively, to genotype the factor V mutation in the Factor V gene, mini-sequencing is conducted in the presence of dCTP and ddATP, ddGTP, and ddTTP.
VSET is also useful for multiplex genotyping, that is the genotyping of multiple single nucleotide polymorphisms. The multiple single nucleotide polymorphism sites may be distributed along one polynucleotides or may be distributed among more than one polynucleotides.
REFERENCES:
patent: 5605798 (1997-02-01), Koster
patent: 5691141 (1997-11-01), Koster
patent: 5885775 (1999-03-01), Haff et al.
patent: 5965363 (1999-10-01), Monforte et al.
patent: 6043031 (2000-03-01), Koster et al.
“A New MALDI-TOF based mini-sequencing assay for genotyping of SNPS” by Sun, et al.,Nucleic Acids Research, 2000, vol. 28, No. 12.
Ausubel FM et al. DNA sequencing. Short Protocols in Molecular Biology. 4th edition, p. 7-1 to 7-4, 1999.*
Sun X et al. A new
Guo Baochuan
Sun Xiyuan
Calfee Halter & Griswold LLP
Campbell Eggerton A.
Chunduru Suryaprabha
Cleveland State University
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