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Mutational analysis

A technology of alleles and probes, applied in the field of analysis of genetic mutations, can solve problems such as no teaching

Active Publication Date: 2014-01-15
EPISTEM
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent does not teach a method to selectively amplify one SNP variation compared to another SNP variation
In addition, it must know the target sequence of interest

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Embodiment 1

[0054] There is a need to define clinical samples to determine the direction of targeted therapy, for example, for the purpose of personalized medicine. This requires rapid and reliable screening for somatic mutations. We show here that the method can be applied to three key somatic mutations in the genes K-RAS, EGFR and BRAF. The method described in this paper utilizes Probes (which provide differential reporter signals depending on whether the probe is single-stranded or double-stranded) and asymmetric PCR (to preferentially amplify one strand of the target sequence). Typical sensitivities of the method are 1-5 copies of the mutant allele, and a SNP:wild-type ratio of greater than 5%. A single assay using a single primer set and probe can detect multiple SNPs within the same probe sequence, for example, a single probe HYB_KRAS_CD12 / 13 detects all 12 mutations in K-RAS codons 12 and 13. Example 1: K-RAS

[0055] figure 1 A partial sequence of the wild-type K-RAS gene is...

Embodiment 2-E

[0062] Example 2 - EGFR

[0063] Figure 10 The EGFR probe sequence (EGFRX18_HYB) for the EGFR exon 18 region is shown. The probe sequence is fully complementary to the wild-type region, whereas there are three possible SNP mutations (2155G>A, 2155G>T and 2156G>C), each of which differs from the probe sequence by a single mismatched base base. Likewise, the EGFRX18_HYB probe is a probe.

[0064] Figure 11 The EGFR probe sequence for EGFR exon 19 is shown (EGFRX19_HYB). Unlike exon 18, where the mutant form is a SNP, exon 19 can carry multiple deletion mutants. Possible missing regions are in Figure 11 in bold, while Figure 12 Exon 19 probes hybridizing to deletion mutants of various lengths are shown. The underlined region of this probe does not hybridize to the target and thus forms a circle. This changes the Tm of the probe:target duplex in much the same way as the presence of a base mismatch in a SNP. The change in Tm depends on the size of the unhybridized r...

Embodiment -B

[0069] Example - BRAF

[0070] The BRAF gene contained multiple potential SNP mutations at amino acid 600: 1799T>A, G or C, and multiple mutations: 1799TG>AT, 1798GT>AA or AG and 1797AGT>GAG. The probe BRAFV600_HYB is fully complementary to a part of the wild-type sequence. The probes and various target sequences are shown in Figure 17 middle.

[0071] Figure 18 The positions of the primer sequences used to amplify the BRAFV600 region are shown; these are outside the probe region. Primer targets are underlined and probe targets are in bold. The figure shows the genome sequence; the probe will be complementary, as will one of the primers.

[0072] These primers were used to amplify various samples, and real-time PCR was used to monitor the hybridization of the probes to the amplified sequences. Melting curves are shown in Figure 19 middle. All samples had a major peak at approximately 52°C, representing the wild-type sequence. The mutant sequence clusters a peak ar...

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Abstract

A method for analysing genetic mutations, and in particular single nucleotide polymorphisms (SNPs) and / or somatic mutations, is described, as well as methods for preferentially amplifying one allelic form compared with another form. The methods use an oligonucleotide probe which hybridises to a first allele with a lower melting temperature (Tm) than that with which it hybridises to a second allele, together with amplification primers which flank the oligonucleotide probe binding site and which bind to the sample with a higher Tm than that of the probe and the first allele. An amplification reaction may be carried out at a temperature such that the probe is preferentially hybridised to the second allele, thereby amplifying the first allele. The amplified sequences may be detected using the same probe as acted as the blocking probe during amplification.

Description

field of invention [0001] The present invention relates to a method for the analysis of genetic mutations, in particular single nucleotide polymorphisms (SNPs) and / or somatic mutations. Aspects of the invention also relate to methods for preferentially amplifying one allelic form over another. Other aspects of the invention relate to nucleic acid probes for use in the analysis of genetic mutations. Background of the invention [0002] Single nucleotide polymorphisms (SNPs) are believed to be of significant potential importance for personalized medicine; it is possible that specific SNPs that result in a single amino acid change may cause changes in a patient's responsiveness to a particular treatment regimen . SNPs may also be important in the development of cancer and other cell proliferative disorders that also have somatic mutations that activate specific oncogenes. [0003] It is important to be able to determine which SNPs are present in a patient. The melting tempe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2549/119C12Q2537/163C12Q2527/107C12Q2537/159C12Q1/6827C12Q2600/156
Inventor 本·科布
Owner EPISTEM
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