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Methods, compositions and kits for detecting allelic variants

An allele and allele-specific technology, applied in the field of rsons BL.D, which can solve problems such as incompatibility and selectivity limitations

Inactive Publication Date: 2011-12-28
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

[0171] Example 1: Tailed primers improve discrimination of allelic variants

[0172] The following examples demonstrate that the use of tail-containing allele-specific primers significantly improves discrimination of allelic variants.

[0173] In traditional AS-PCR, discrimination of 3' nucleotide mismatches is highly dependent on the sequence surrounding the SNP and the nature of the allele. ΔC between amplification reactions of matched and mismatched primers t is changing. 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.

[0174] Assays were performed using the general experimental design and reaction conditions described above (except that blocking probes were not included), using 0.5 ng / uL of genomic DNA comprising the hsv11711720 SNP containing three alleles as nucleic acid template One of (A, C o...

Embodiment 2

[0184] Example 2: Low primer concentrations improve discrimination of allelic variants

[0185] 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 for the sequences indicated in -D.

[0186] 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.

[0187] Table 5: Assay results using different concentrations of tailed allele-specific primers

[0188] CV11201742

Embodiment 3

[0189] Example 3: Primers designed with reduced Tm improve discrimination of allelic variants

[0190] 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 11A Design assay primers and probes for the sequences indicated in -D.

[0191] 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.

[0192] Table 6: [Delta]Ct values ​​using tailed ASPs with lower Tm (-53°C) or higher Tm (-57°C).

[...

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PUM

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Abstract

In some embodiments, the present invention generally relates to compositions, methods and kits for distinguishing sequence differences between different alleles. More specifically, in some embodiments, the present invention provides methods for quantifying rare (e.g., mutant) alleles in samples containing abundant (e.g., wild-type) allelic variants with high specificity and selectivity. Compositions, methods and kits for genetic variants such as SNP or nucleotide (NT) insertion or deletion. In particular, in some embodiments, the present invention relates to a highly selective method for detecting mutations known as competitive allele-specific TaqMan PCR ("cast-PCR").

Description

[0001] Cross References to Related Applications [0002] This application is required under 35 U.S.C. 119 of U.S. Provisional Application Nos. 61 / 138,521 filed December 17, 2009, 61 / 258,582 filed November 5, 2009, 61 / 253,501 filed October 20, 2009, 2009 Priority of 61 / 251,623 filed October 14, 61 / 186,775 filed June 12, 2009, and 61 / 164,230 filed March 27, 2009, all of which are incorporated herein by reference in their entirety. 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C07H21/04C12Q2561/113C12Q2561/101C12Q2537/161C12Q1/686C12Q1/6883C12Q1/6886C12Q2600/156C12Q2600/172
Inventor C·陈R·谭
Owner LIFE TECH CORP
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