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Methods and products related to genotyping and DNA analysis

a genotyping and dna technology, applied in the field of methods and products related to genotyping, can solve the problems of high cost, difficult identification of complex gene mutations contributing to diabetes disorders, and high cost of southern blotting, and achieve the effect of high throughpu

Inactive Publication Date: 2009-04-16
MASSACHUSETTS INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0040]According to another aspect of the invention, a kit is provided. The kit includes a container housing a set of PCR primers for reducing the complexity of a genome, and a container housing a set of SNP-ASOs. The SNPs which correspond to the SNP-ASOs of the kit are preferably present within a RCG made using the PCR primers of the kit with a frequency of at least 50%.

Problems solved by technology

Although substantial progress has been made in identifying the genetic basis of many human diseases, current methodologies used to develop this information are limited by prohibitive costs and the extensive amount of work required to obtain genotype information from large sample populations.
These limitations make identification of complex gene mutations contributing to disorders such as diabetes extremely difficult.
Southern blotting is both expensive and time-consuming when performed on large numbers of samples, such as those required to identify a complex genotype associated with a particular phenotype.
One problem with this type of analysis is that “stuttering” tends to occur, causing a smeared result and making the data difficult to interpret and score.
The gene chip method is also expensive and time-intensive.

Method used

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  • Methods and products related to genotyping and DNA analysis
  • Methods and products related to genotyping and DNA analysis
  • Methods and products related to genotyping and DNA analysis

Examples

Experimental program
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Effect test

example 1

Identification and Isolation of SNPs

[0196]High allele frequency SNPs are estimated to occur in the human genome once every kilobase or less (Cooper et al., 1985). A method for identifying these SNPs is illustrated in FIG. 1. As shown in FIG. 1, inter-Alu PCR was performed on genomes isolated from three unrelated individuals. The PCR products were cloned, and a mini library was made for each of the 3 individuals. The library clone inserts were PCR-amplified and spotted on nylon filters. Clones were matched by hybridization into two sets of identical clones from each individual, for a total of 6 clones per matched clone set. These sets of clones were sequenced, and the sequences were compared in order to identify SNPs. This method of identifying SNPs has several advantages over the prior art PCR amplification methods. For instance, a higher quality sequence is obtained from cloned DNA than is obtained from cycle sequencing of PCR products. Additionally, every sequence represents a spe...

example 2

Allele-Specific Oligonucleotide Hybridization to Alu PCR SNPs

Methods

[0205]Inter-Alu PCR was performed using genomic DNA obtained from 136 members of 8 CEPH families (numbers 102, 884, 1331, 1332, 1347, 1362, 1413, and 1416) using the 8C Alu primer, as described above. The products from these reactions were denatured by alkali treatment (10-fold addition of 0.5 M NaOH, 2.0 M NaCl, 25 mM EDTA) and dot blotted onto multiple Hybond™ N+ filters (Amersham) using a 96-well dot blot apparatus (Schleicher and Schull). For each SNP, a set of two allele-specific oligonucleotides consisting of two 17-residue oligonucleotides centered on the polymorphic nucleotide residue were synthesized. Each filter was hybridized with 1 picomole 32P-kinase labeled allele-specific oligonucleotides and a 50-fold excess of non-labeled competitor oligonucleotide complementary to the opposite allele (Shuber et al., 1993). Hybridizations were carried out overnight at 52° C. in 10 mL TMAC buffer 3.0 M TMAC, 0.6% SDS...

example 3

Confirmation of SNP Identity

[0209]Allele-specific oligonucleotides are synthesized based on standard protocols (Shuber et al., 1997). Briefly, polynucleotides of 17 bases centering on the polymorphic site are synthesized for each allele of a SNP. Hybridization with DNA dots of IRS or DOP-PCR products affixed to a membrane were performed, followed by hybridization to end labeled allele-specific oligonucleotides under TMAC buffer conditions. These conditions are known to equalize the contribution of AT and GC base pairs to melting temperature, thereby providing a uniform temperature for hybridization of allele-specific oligonucleotides independent of nucleotide composition.

[0210]Using this methodology, genotypes of CEPH progenitors and their offspring are determined. The Mendelian segregation of each SNP marker confirms its identity as a SNP marker and accrued estimate of its relative allele frequency, hence, its likely usefulness as a genetic marker. Markers which yield complex segre...

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Abstract

The invention encompasses methods and products related to genotyping. The method of genotyping of the invention is based on the use of single nucleotide polymorphisms (SNPs) to perform high throughput genome scans. The high throughput method can be performed by hybridizing SNP allele-specific oligonucleotides and a reduced complexity genome (RCG). The invention also relates to methods of preparing the SNP specific oligonucleotides and RCGs, methods of fingerprinting, determining allele frequency for a SNP, characterizing tumors, generating a genomic classification code for a genome, identifying previously unknown SNPs, and related compositions and kits.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 676,154, filed on Sep. 29, 2003, pending, which claims priority to U.S. application Ser. No. 09 / 404,912, filed on Sep. 24, 1999, now U.S. Pat. No. 6,703,228, granted Mar. 9, 2004, which claims priority to U.S. Provisional Application No. 60 / 101,757, filed Sep. 25, 1998, the entire contents of which is hereby incorporated by reference.GOVERNMENT SUPPORT[0002]The present invention was supported in part by a grant from the United States National Institutes of Health under contract / grant number 5-R01-HG00299-18; the National Cancer Institute of Canada under contract / grant #009645; 007477; National Research Foundation DHHS, NIH, NCI, 5 F32 CA73118-03 and NIH Predoctoring Grant T32 GM07287. The U.S. Government has rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates to methods and products associated with genotyping. In particular, the invention relates to methods of d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G06F17/30G06F19/00G16B20/20G16B30/00
CPCC12Q1/6827C12Q1/6886C12Q2600/172G06F19/18G06F19/22C12Q1/686C12Q1/6837C12Q2531/113C12Q2525/179C12Q2535/131G16B20/00G16B30/00G16B20/20
Inventor LANDERS, JOHNJORDAN KLANDERMAN, BARBARAHOUSMAN, DAVID E.CHAREST, ALAIN
Owner MASSACHUSETTS INST OF TECH
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