Methods and kits for linking polymorphic sequences to expanded repeat mutations
a polymorphic sequence and expanded repeat technology, applied in the field of methods and kits for linking polymorphic sequences to expanded repeat mutations, can solve the problems of not disclosing the use of reverse transcription, unfavorable patient safety, and high care cos
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example 1
[0111]RNA-Isolation and Reverse Transcription Reaction.
[0112]Applicants analyzed the CAG-repeat sequences in the Huntington's disease gene in the mRNA obtained from a patient's cells using the following allele-specific reverse transcription reaction. The subject SNP sites in the Huntington's disease gene (IT15) are designated using the identification number provided by the National Center for Biotechnology Information (NCBI) database, accessible at: http: / / www.ncbi.nlm.nih.gov / entrez / query.fcgi?db=snp.
[0113]Development of Allele-Specific Reverse Transcription.
[0114]Donor-1 (a Caucasian female who was 28 years old at the time of cell collection and whose mother was diagnosed with HD at the age 49) was determined to be heterozygous (adenine versus cytosine) at SNP site rs363125. In order to design an allele-specific RNA interference-based therapy for donor-1, it is necessary to determine which of these SNP sequences is associated with the expanded CAG repeat mutation that is located a...
example 2
[0119]Development of SNP-Specific Real Time PCR Assays.
[0120]To be able to verify that allele-specific suppression of HD mRNA is occurring in cells, it is necessary to be able to quantify the amount of HD mRNA corresponding to each allele individually. Molecular beacons are synthetic oligonucleotide probes that have a fluorophore and a quencher covalently linked to the respective ends of the oligo. In solution, the beacon adopts a hairpin conformation, causing the fluorophore to be quenched. However, upon hybridization with complementary DNA in a PCR reaction, the hairpin conformation is lost and fluorescence from the fluorophore can be detected. Beacons can be constructed such that as little as a single nucleotide mismatch between the beacon and the complementary DNA is sufficient for the probe to be more stable in its self-annealed state than in the probe-cDNA hybrid. The inventors designed two such beacons corresponding to the two allelic variants of SNP rs363125, for the A allel...
example 3
[0122]Allele-Specific Suppression of Huntingtin mRNA.
[0123]In one set of experiments, fibroblasts were cultured in 25 cm2 culture flasks (Nunc) as described, but without the addition of PSN antibiotics and Fungizone. Lipofectamine 2000 (Invitrogen) was used to conduct siRNA transfection at three different conditions: 1) mock transfection (n=8); 2) transfection with scrambled siRNA (Ambion) (n=4); and 3) transfection with siRNA sequence 5′-GAAGUACUGUCCCCAUCUCdTdT-3′, SEQ ID NO: 228 and its complementary strand SEQ ID NO: 229, (Ambion) (n=7) at a concentration of 100 nM. This siRNA has a guanine located at position 16 relative to the 3′ end of the complementary region of the target huntingtin mRNA, providing specificity for the allele containing cytosine at the SNP position. A parallel cell culture was transfected with a fluorescently labeled Block-It siRNA (Invitrogen) to verify efficiency of Lipofectamine 2000 transfection. The cells were incubated overnight at 37° C., 5% CO2 and ma...
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