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New insights into the pathogenic mechanisms associated with CNVs: duplication of 17p13.3, mirror effect in 16p11.2 and recessive phenotype in 22q11.22

von Dott. Mafalda Mucciolo

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Statistik und Sichtungsnachweis dieser Seite findet sich am Artikelende
[1.] Mmu/Fragment 025 01 - Diskussion
Zuletzt bearbeitet: 2016-02-07 14:52:21 Hindemith
Fragment, Gesichtet, KomplettPlagiat, Mmu, Papa 2010, SMWFragment, Schutzlevel sysop

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KomplettPlagiat
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SleepyHollow02
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Untersuchte Arbeit:
Seite: 25, Zeilen: 1 ff. (komplett)
Quelle: Papa 2010
Seite(n): 30 f., Zeilen: 30: 8 ff.; 31: 1 ff.
3.3 Real-time quantitative PCR

Some aCGH data were confirmed by Real-time Quantitative PCR experiments. To design adequate probes in different regions of the human genome, we used an TaqMan Gene Expression Assays by design which provides pre-designed primers-probe set for real-time PCR experiments (Applied Biosystems, https://products.appliedbiosystems.com). PCR was carried out using an ABI prism 7000 (Applied Biosystems) in a 96-well optical plate with a final reaction volume of 50 μl. A total of 100 ng (10 μl) was dispensed in each of the four sample wells for quadruplicate reactions. Thermal cycling conditions included a pre-run of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 sec and 60°C for 1 min according to the TaqMan Universal PCR Protocol (ABI). The TaqMan Universal PCR Master Mix and Microamp reaction tubes were supplied by Applied Biosystems. The starting copy number of the unknown samples was determined using the comparative Ct method as previously described (Ariani 2004).

3.4 Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA analysis was performed according to the provider’s protocol with a specifically designed set of probes for testing critical regions in DiGeorge syndrome (SALSA P023 kit; MRC-Holland, Amsterdam, Netherlands; http://www.mrc-holland.com), 1p-deletion syndrome, Williams syndrome, Smith-Magenis syndrome, Miller-Dieker syndrome, DiGeorge syndrome, Prader-Willi syndrome, Alagille syndrome, Saethre-Chotzen syndrome, Sotos syndrome: (SALSA P064B MR1 kit) and subtelomere regions (SALSA P036D subtelomeric primer kit). The ligation products were amplified by PCR using the common primer set with the 6-FAM label distributed by the supplier. Briefly, 100 ng of genomic DNA was diluted with TE buffer to 5 μl, denatured at 98°C for 5 minutes and hybridized with SALSA Probe-mix at 60°C overnight. Ligase-65 mix was then added and ligation was performed at 54°C for 15 minutes. The ligase was successively inactivated by heat, 98°C for 5 minutes. PCR reaction was performed in a 50 μl volume. Primers, dNTP and polymerase were added and amplification was carried out for 35 cycles (30 seconds [at 95°C, 30 seconds at 60°C and 60 seconds at 72°C).]


2. Ariani, F., et al., Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat, 2004. 24(2): p. 172-7.

3.3 Real-time quantitative PCR

Some aCGH data were confirmed by Real-time Quantitative PCR experiments. To design adequate probes in different regions of the human genome, we used an TaqMan Gene Expression Assays by design which provides pre-designed primers-probe set for real-time PCR experiments (Applied Biosystems, https://products.appliedbiosystems.com) PCR was carried out using an ABI prism 7000 (Applied Biosystems) in a 96-well optical plate with a final reaction volume of 50 μl. A total of 100 ng (10 μl) was dispensed in each of the four sample wells for quadruplicate reactions. Thermal cycling conditions included a prerun of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 sec and 60°C for 1 min according to the TaqMan Universal PCR Protocol (ABI). The TaqMan Universal PCR Master Mix and Microamp reaction tubes were supplied by Applied Biosystems. The starting copy number of the unknown samples was determined using the comparative Ct method as previously described. [95]

[page 31:]

3.4 Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA analysis was performed according to the provider’s protocol with a specifically designed set of probes for testing critical regions in DiGeorge syndrome (SALSA P023 kit; MRC-Holland, Amsterdam, Netherlands; http://www.mrc-holland.com), 1p-deletion syndrome, Williams syndrome, Smith- Magenis syndrome, Miller-Dieker syndrome, DiGeorge syndrome, Prader-Willi syndrome, Alagille syndrome, Saethre-Chotzen syndrome, Sotos syndrome: (SALSA P064B MR1 kit) and subtelomere regions (SALSA P036D subtelomeric primer kit). The ligation products were amplified by PCR using the common primer set with the 6-FAM label distributed by the supplier. Briefly, 100 ng of genomic DNA was diluted with TE buffer to 5 μl, denatured at 98°C for 5 minutes and hybridized with SALSA Probe-mix at 60°C overnight. Ligase-65 mix was then added and ligation was performed at 54°C for 15 minutes. The ligase was successively inactivated by heat, 98°C for 5 minutes. PCR reaction was performed in a 50 μl volume. Primers, dNTP and polymerase were added and amplification was carried out for 35 cycles (30 seconds at 95°C, 30 seconds at 60°C and 60 seconds at 72°C).


95. Ariani, F., et al., Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat, 2004. 24(2): p. 172-7.

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