Method for determining polynucleotide sequence variations

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S091200, C536S022100

Reexamination Certificate

active

06322988

ABSTRACT:

BACKGROUND
Individual DNA sequence variations in the human genome are known to directly cause specific diseases or conditions, or to predispose certain individuals to specific diseases or conditions. Such variations also modulate the severity or progression of many diseases. Additionally, DNA sequence variations between populations. Therefore, determining DNA sequence variations in the human genome is useful for making accurate diagnoses, for finding suitable therapies, and for understanding the relationship between genome variations and environmental factors in the pathogenesis of diseases and prevalence of conditions.
There are several types of DNA sequence variations in the human genome. These variations include insertions, deletions and copy number differences of repeated sequences. The most common DNA sequence variations in the human genome, however, are single base pair substitutions. These are referred to as single nucleotide polymorphisms (SNPs) when the variant allele has a population frequency of at least 1%.
SNPs are particularly useful in studying the relationship between DNA sequence variations and human diseases and conditions because SNPs are stable, occur frequently and have lower mutation rates than other genome variations such as repeating sequences. In addition, methods for detecting SNPs are more amenable to being automated and used for large-scale studies than methods for detecting other, less common DNA sequence variations.
A number of methods have been developed which can locate or identify SNPs. These methods include dideoxy fingerprinting (ddF), fluorescently labeled ddF, denaturation fingerprinting (DnF1R and DnF2R), single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis, heteroduplex analysis, RNase cleavage, chemical cleavage, hybridization sequencing using arrays and direct DNA sequencing.
The known methods for locating or identifying SNPs are associated with certain disadvantages. For example, some known methods do not identify the specific base changes or the precise location of these base changes within a sequence. Other known methods are not amenable to analyzing many samples simultaneously or to analyzing pooled samples. Still other known methods require different analytical conditions for the detection of each variation. Additionally, some known methods cannot be used to quantify known SNPs in genotyping assays. Further, many known methods have excessive limitations in throughput.
Thus, there is a need for a new method to determine the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, including the presence of an SNP in the genome of a human individual. Preferably, the method could determine the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide in a pooled sample. Additionally preferably, the method could determine whether two or more variations reside on the same or different alleles in an individual, and could be used to determine the frequency of occurrence of the variation in a population. Further preferably, the method could screen large numbers of samples at a time with a high degree of accuracy.
SUMMARY
In one embodiment, there is provided a method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide. The method comprises, first, providing a sample of the first polynucleotide and selecting a region of the first polynucleotide potentially containing the variation. Then, the selected region is subjected to a template producing amplification reaction to produce a plurality of double stranded polynucleotide templates which include the selected region. Next, a family of labeled, linear polynucleotide fragments is produced from both strands of the template simultaneously by a fragment producing reaction using a set of primers. Each of the family of fragments is terminated by a terminator at the 3′ end of the fragment. The family of fragments includes at least one fragment terminating at each possible base, represented by the terminator, of that portion of both template strands flanked by the primers. Then, the locations and identities of at least some of the bases in the selected region of the first polynucleotide are determined using the labels present in the fragments. Next, the location and identity of the bases determined is compared with the location and identity of bases from a second polynucleotide, thereby identifying the presence and identity of a variation in a nucleotide sequence between the selected region of the first polynucleotide and a corresponding region of the second polynucleotide.
DESCRIPTION
The present invention includes a method for determining the presence, location or identity, or a combination of these, of one or more polynucleotide sequence differences between at least two polynucleotides. Among other uses, the present method can locate and identify single nucleotide polymorphisms present in the human genome. Further, the present method can discover previously unidentified genome variations between individuals, between an individual and a population, and between populations. Also, the present method can determine the frequency or distribution of genome variations within populations. Additionally, the present method can relate specific genome variations found in a population to specific phenotypes within that population. Still further, the present method can determine the allelic distribution of genome variations in individuals and populations.
More specifically, the present method of the present invention can provide the following types of information on polynucleotide sequence variation between two polynucleotides. First, the present method can identify the position of all the nucleotides in a selected region of a first polynucleotide that are different from one or more additional polynucleotides. Second, the present method can identify which nucleotide has replaced another nucleotide in a polynucleotide. Third, the present method can determine the proportion of the polynucleotide molecules that have each of the nucleotide changes that can occur at a given location in the sequence. Fourth, where two different polynucleotides have a plurality of nucleotide differences, the present method can provide information on which differences occur together.
The present method has several combined advantages over known methods. Generally, the present method provides more types of information, is more widely applicable and is simpler to perform. Particularly advantageous, the present method is a single technology that can simultaneously identify and quantitate known and unknown variations and determine the locations, identities and frequencies of all variations between two populations of polynucleotides. Additionally, the present method can determine whether two or more genetic variations reside on the same or different alleles in an individual, and can be used to determine the frequency of occurrence of the variation in a population.
Further, the present method can be used on any type of polynucleotide, from any source. In addition to determining the location and identity of SNPs, the present method can be used to determine the presence and type of polynucleotide variations including substitutions, deletions, insertions, expansions and contractions involving multiple nucleotides, and truncated or chimeric molecules. Further, the present method can identify alterations in the relative copy number of sequences in diploid organisms that involve the loss of one copy of a polynucleotide such as loss of heterozygosity, or that involve the gain of additional copies of a polynucleotide such as conditions in which extra copies of chromosomes are present.
Additionally, in population studies, the present method can be used to determine the frequencies of each polynucleotide variation by analysis of a single pooled sample that is composed of samples taken from multipl

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