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Methods, compositions, and kits for detecting allelic variants

An allele, allele-specific technology, applied in the field of detection of allelic variants, compositions and kits, can solve the problems of limited selectivity and inappropriateness

Active Publication Date: 2012-04-25
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as with AS-PCR, selectivity using this method is limited and it is not suitable for detection of rare (≥1 in 1,000) alleles or mutations in mixed samples

Method used

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  • Methods, compositions, and kits for detecting allelic variants
  • Methods, compositions, and kits for detecting allelic variants
  • Methods, compositions, and kits for detecting allelic variants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0240] Example 1: Tailed primers improve discrimination of allelic variants

[0241] The following examples demonstrate that the use of tail-containing allele-specific primers significantly improves discrimination of allelic variants.

[0242] In traditional AS-PCR, discrimination of 3' nucleotide mismatches is highly dependent on the sequence surrounding the SNP and the nature of the allele. The ΔCt varies between amplification reactions with matched and mismatched primers. To improve the discrimination between the amplification of matched and mismatched sequences, allele-specific primers were designed to contain a tail at their 5' end and then tested for their suitability in the AS-PCR assay.

[0243] Assays were performed using the general experimental design and reaction conditions described above (except that blocking probes were not included and tailed or non-tailed allele-specific primers were added), using 0.5 ng / uL of genomic DNA as A nucleic acid template, the geno...

Embodiment 2

[0255] Example 2: Low primer concentrations improve discrimination of allelic variants

[0256] Using the general experimental design and reaction conditions described above, in the presence of 1,000,000 copies of plasmid DNA containing multiple SNP target sequences (see Table 1) and 200 nM or 800 nM tailed ASP (as indicated), To measure. according to Figure 11A Design assay primers and probes with the indicated sequences.

[0257] The effect of tailed ASP concentration on discrimination of allelic variants is summarized in Table 5. The ΔCt between amplification reactions of matched and mismatched primers demonstrates that lower tailed ASP concentrations improve discrimination of allelic variants.

[0258] Table 5: Assay results using different concentrations of tailed allele-specific primers

[0259]

Embodiment 3

[0260] Example 3: Primers designed with reduced Tm improve discrimination of allelic variants

[0261] Using the general experimental design and reaction conditions as described above, tailing with a higher Tm (~57°C) was used in the presence of 1,000,000 copies of plasmid DNA containing multiple SNP target sequences (see Table 1). or tailed ASP with a lower Tm (~53°C). according to Figure 11B Design assay primers and probes for sequences indicated in -E (for higher Tm ASPs, see Figure 11B ; for lower Tm ASP, see Figure 11C ).

[0262] The effect of Tm of allele-specific primers on discrimination of allelic variants is summarized in Table 6. The ΔCt of allele-specific primers with lower Tm was significantly higher than that of allele-specific primers with higher Tm. Allele-specific primers designed with reduced Tm improved discrimination of the allelic variants by up to 118-fold in some cases, with an average of about 13-fold difference.

[0263] Table 6: [Delta]Ct va...

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PUM

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Abstract

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR ('cast-PCR').

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of U.S. Application Serial No. 12 / 641,321, filed December 17, 2009, and pursuant to 35 U.S.C. 119, 61 / 258,582, filed November 5, 2009, 61 / 253,501, 61 / 251,623 filed October 14, 2009, 61 / 186,775 filed June 12, 2009, and 61 / 164,230 filed March 27, 2009, all of which are incorporated by reference in their entirety into this article. Background technique [0003] Single Nucleotide Polymorphism (SNP) is the most common type of genetic diversity in the human genome, and its frequency of occurrence in human genomic DNA is: about 1 SNP in 1,000 nucleotides, or less (Kwok, P-Y, Ann Rev Genom Hum Genet 2001, 2:235-258). SNPs have been implicated in genetic disorders, susceptibility to different diseases, predisposition to adverse reactions to drugs, and are used in forensic research. Therefore, the detection of SNPs (or rare mutations) offers great potential for early diagnosis of dise...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2561/113C12Q2561/101C12Q2537/161C12Q2525/186C12Q2565/107C12Q2535/125C12Q1/6883C12Q2600/156C12Q1/686
Inventor C·陈R·谭
Owner LIFE TECH CORP
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