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Methods and probes for identifying a nucleotide sequence

a nucleotide sequence and probe technology, applied in the field of molecular biology, can solve the problems of reducing the number of probes required for the application, reducing the number of probes, and increasing the cost of the process

Inactive Publication Date: 2008-10-09
SIMONS HAPLOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for identifying a set of target nucleotide sequences that can be used to identify members of a group of related nucleotide sequences. This method involves dividing the nucleotide sequence of each member of the group into a plurality of subsequences, at least two of which overlap. This approach allows for the identification of probes that can accurately identify a member of the group. The method can be automated and is useful for analyzing genes with a high number of alleles or polymorphic sites. The set of probes generated can be directed to multi-exon coverage and can provide complete allele assignment. The invention also provides a computer executable program for executing the methods described herein."

Problems solved by technology

While automated sequencing has been possible for some years, the process is still time intensive and expensive.
The disadvantage of this method is that where there is no endonuclease specific for each and every SNP in the range of alleles, then all alleles will not be identifiable by RFLP.
This is often the case, and so use of RFLP is significantly limited.
A problem of oligonucleotide probe-based methods is that to definitively ascribe a genotype it may be necessary to use a very large number of probes.
Generating such a large number of different oligonucleotide probes, and then assessing the ability of each probe to hybridise to a test sample, is clearly a significant burden.
Furthermore, previously unrecognised alleles continue to be discovered thereby exacerbating the problem of providing a probe set capable of resolving an individual's HLA type.
A problem with this approach is that it is not systematic, and it is necessary for a human to judiciously design the probes.
Given the real possibility of error in this process it remains an uncertainty whether the probe set will identify all alleles at the end of the probe design process.
A further problem with the method provided by Guo et al is that it is necessary to include SNP sites over the length of the probe.

Method used

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  • Methods and probes for identifying a nucleotide sequence

Examples

Experimental program
Comparison scheme
Effect test

example 1

Identification of Oligonucleotide Probe Set for Definitive Genotyping of the HLA-DRB Locus

Outline of Protocol

[0098]The DRB locus of HLA was analyzed by the present methods to identify a probe set capable of identifying any known allele of the locus. The DRB locus has the following known alleles:

DRB1*010101, DRB1*010102, DRB1*010103, DRB1*010201, DRB1*010202, DRB1*010203, DRB1*010204, DRB1*0103, DRB1*0104, DRB1*0105, DRB1*0106, DRB1*0107, DRB1*0108, DRB1*0109, DRB1*0110, DRB1*0111, DRB1*0112, DRB1*0113, DRB1*030101, DRB1*030102, DRB1*030201, DRB1*030202, DRB1*0303, DRB1*0304, DRB1*030501, DRB1*030502, DRB1*0306, DRB1*0307, DRB1*0308, DRB1*0309, DRB1*0310, DRB1*0311, DRB1*0312, DRB1*0313, DRB1*0314, DRB1*0315, DRB1*0316, DRB1*0317, DRB1*0318, DRB1*0319, DRB1*0320, DRB1*0321, DRB1*0322, DRB1*0323, DRB1*0324, DRB1*0325, DRB1*0326, DRB1*0327, DRB1*0328, DRB1*040101, DRB1*040102, DRB1*0402, DRB1*040301, DRB1*040302, DRB1*0404, DRB1*040501, DRB1*040502, DRB1*040503, DRB1*040504, DRB1*0406,...

example 2

Production of Microarray Chip

[0105]The 5,500 target nucleotide sequences in the pool are synthesized directly onto a microarray chip by Affymetrix Inc who provide a custom gene chip array service.

example 3

Use of Probes to Assign Identify DRB Allele for an Individual

Patient Sample.

[0106]DNA extraction of peripheral blood or buccal smear is standard practice. Approx. 1,000 ng of DNA is recommended for microarray assay.

Long PCR.

[0107]Primers can be located in introns, exons or a combination. For instance, for HLA-DRB typing, primers are selected upstream in intron 1, and downstream in exon 6. The amplicon is approx. 5.1 kb. The disadvantage of using intron sequences as primer sites is that there is usually less sequence data, and absence of data corresponding to exon alleles, than for corresponding exon sequence. For HLA-DRB, published data provides sufficient intron 1 data for primer selection. However, even in this case, further sequencing is near certain to reveal new SNPs. If they occur in the primer sequence, it can be expected to complicate amplification of sequences bearing that new variant. The alternative is to utilise exon sequences since these have been more extensively studi...

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Abstract

The present invention provides a method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap. The method is useful in generating probe sets capable of assigning alleles at HLA or KIR loci.

Description

FIELD OF THE INVENTION[0001]The present invention is directed to the field of molecular biology. More specifically the invention is directed to methods for generating oligonucleotide probes and uses thereof in identifying members of a group of related nucleotide sequences. The methods and probes may be used in identifying an allele of a gene in an individual.BACKGROUND TO THE INVENTION[0002]The Human Genome Project has highlighted the importance of single nucleotide polymorphisms (SNPs) in the genome. These polymorphisms occur on average every 100 to 300 bases throughout the genome. While the genes of all humans are known to be more than 99% identical, it is presence of SNPs that provide a major component of genetic diversity in a species. Different alleles of a gene can confer very different phenotypes on an individual including characteristics as diverse as disease resistance, the ability to respond to a pharmaceutical compound, sporting ability and the like.[0003]Plant genomes al...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/06G01N33/50C12Q1/68C07H21/00G16B25/20G16B30/00
CPCC12N15/1093C12Q1/6881Y10T436/143333G06F19/22G06F19/20C12Q2600/156G16B25/00G16B30/00G16B25/20
Inventor SIMONS, MALCOLM JAMES
Owner SIMONS HAPLOMICS