Detection of STRP, such as fragile X syndrome

An X-chromosome and syndrome technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of limited capacity and inability to produce enough PCR products, etc.

Inactive Publication Date: 2007-03-07
BIOCEPT INC
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Problems solved by technology

[0011] However, these methods are limited in their ability to determine the number of CGG repeats to provide an accurate diagnosis
The reason is usually the difficulty of PCR amplifying the CGG repeat region and not generating enough PCR product for accurate gel electrophoresis analysis (a large amount of PCR product is required for detection)

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  • Detection of STRP, such as fragile X syndrome

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Embodiment

[0100] PCR was performed in a total volume of 50 ul containing 10 ng of genomic DNA, 1 uM each of the primers targeting the selected nucleic acid segment of the X chromosome where the FRAXA sequence is located. The selected segment includes the complete CGG repeat portion and the 3' internal standard segment adjacent to it. Roche's GC-Rich PCR system was used. The FRAXA forward and reverse primers used were oligonucleotides having the nucleotide base sequences shown in SEQ ID NO: 1 and 2 (see Table). The length of the forward primer is 21 nucleotides, and the length of the reverse primer is 27 nucleotides. They span a total gene segment that is at least 254 nucleotides in length on the "normal" X chromosome. The forward PCR primer is 5' biotinylated and the reverse primer is 5' phosphorylated. The PCR temperature cycling conditions used were: 95°C for 2 minutes, followed by 25 cycles of 95°C for 1.5 minutes, 56°C for 1 minute, and 72°C for 2 minutes. The final extension wa...

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Abstract

Methods for detecting a short tandem repeat polymorphism (STRP), such as fragile X syndrome, wherein PCR is used to amplify nucleic acid along the chromosome in the genomic DNA which includes all of the STRs of interest plus a substantial contiguous segment of the nucleic acid adjacent to the STRs. Single-stranded product is then obtained, and colorimetric-labeled oligonucleotides which target for (i) STRs and (ii) the contiguous DNA segment are hybridized with this single-stranded product which is then bound to a solid phase and separated from the remainder of the target material. The labeled oligonucleotide target material is recovered by treatment with base and then hybridized to a microarray having a plurality of spots containing suitable oligonucleotide probes complementary thereto. Following hybridization, colorimetric intensities of the hybridized labeled target material present at specific spots on the microarray are measured to obtain individual values which are compared with results from known control samples to accurately quantify the number of STRs in the region of interest of the DNA being analyzed.

Description

field of invention [0001] The present invention relates generally to diagnostic tests for hereditary or sporadic genetic defects in adults, fetuses and embryos, and more particularly to the detection of diseases or defects caused by short tandem repeats (STRs), and more particularly to the detection of diseases or defects that cause Fragile X Detection of genetic defects in Fragile X syndrome. These STRP assays use polymerase chain reaction (PCR), which is then hybridized to a microarray and analyzed. Background of the invention [0002] Eukaryotic DNA has very short tandem repeats of simple sequences called short tandem repeat polymorphisms (STRPs). Repeat polymorphisms include dinucleotide, trinucleotide and tetranucleotide repeats. Trinucleotide and tetranucleotide repeats are repeats of 3 nucleotides and 4 nucleotides. An increasing number of diseases are known to be associated with expansion of trinucleotide STRs (Trottier, Y. et al., Current Biology 3:783-786 (1993)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/156C12Q1/6883C12Q1/6827
Inventor S·韩
Owner BIOCEPT INC
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