The ZMNH Core Facility Bioanalytics
The Service Facility Bioanalytics at the ZMNH is equipped with an ABI PRISM Genetic Analyzer and two HT 7900 RT-PCR devices. The here offered service includes the implementation of Sanger sequence reactions, the subsequent purification and analysis with an ABI PRISM Genetic Analyzer and, optionally, the accomplishment of fragment analyses. For real time PCR experiments two 7900 HT devices are available. As service - typically for transgenic mice - genotype determinations are conducted using High Resolution Melting, RT-PCR and Copy Number Variation assays. The respective assays can be designed and established if desired.
The sample DNA should be as clean as possible: The quality of the DNA directly correlates with the quality of the sequence. The submitted samples should contain approximately 500 ng DNA (for average sized plasmids), 5 - 10 pmol primer and water in a final volume of 8 ul. Please make sure that the sample does not contain any EDTA if possible! The results are received electronically; in addition, printouts are offered on request.
In cooperation with the client assays (z. Ex. Spectratyping) can be designed and carried out; the billing is based on the expended material costs.
RT - PCR
The following applications can be carried out with our equipment:
1. absolute quantification (includes CNV assays)
2. relative quantification
3. end-point analysis: allelic discrimination, SNP detection Procedure:
Please apply in time for the planned runs and leave your phone number.
Please bring your own "Optical Cover Compression Pads"!
Please do not forget to remove the microtiter plate from the unit, after the end of the run.
HRM and CNV
The HRM (High Resolution Melting) analysis discriminates between DNA sequences based on differences in their composition, their length, their GC - content, or strand complementarity, that is, if for example two mouse lines differ in a particular gene because of the presence of a nucleotide exchange, a deletion or insertion, this will be detected by amplification of the gene’s relevant segment and subsequent melting curve analysis.
The inbred strain C57BL / 6J is the reference strain for most behavioral and physiological phenotypes. However, since all ES cell clones derived from the International Knockout Mouse Consortium (IKMC) go back to the C57BL / 6N sub tribe, their genetic background must be analyzed before a thorough analysis of the mice can be started.
In C57BL/6N -Sub tribe present mutations / variations that are analyzed here as a service:
- Crb1rd8 Chang et al., and Mattapallil et al.
- Cyfip2B6N/– Kumar et al.
- Nnt, 11 kb deletion in J-sub tribe Mekada et al.
Another way for molecular genetic characterization of mouse strains lies in the determination of copy number variations (CNV), that is larger DNA sections that may be present in different copy numbers. Likewise using such assays, it is possible to achieve an assessment whether a particular transgene is present as a single segment, as in the heterozygous case, or as two segments, as in the homozygote situation. Appropriate assays can be designed and established if desired.
Chang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. Retinal degeneration mutants in the mouse. Vision Res. 2002; 42: 517 - 525
Mary J. Mattapallil, Eric F. Wawrousek, Chi-Chao Chan, Hui Zhao, Jayeeta Roychoudhury, Thomas A. Ferguson, and Rachel R. Caspi. The Rd8 Mutation of the Crb1 Gene Is Present in Vendor Lines of C57BL/6N Mice and Embryonic Stem Cells, and Confounds Ocular Induced Mutant Phenotype. Investigative Ophthalmology & Visual Science, May 2012, Vol. 53, No. 6
Vivek Kumar, Kyungin Kim, Chryshanthi Joseph, Saïd Kourrich, Seung-Hee Yoo1, Hung Chung Huang1, Martha H. Vitaterna, Fernando Pardo-Manuel de Villena, Gary Churchill, Antonello Bonci, Joseph S. Takahashi. C57BL/6N Mutation in Cytoplasmic FMRP interacting protein 2 Regulates Cocaine Response. Science, December 2013, Vol. 342 no. 6165 pp. 1508-1512.
Mekada K, Abe K, Murakami A, Nakamura S, Nakata H, Moriwaki K, Obata Y, Yoshiki A. Genetic differences among C57BL/6 substrains. Exp Anim. 2009 Apr;58(2):141-9.
The Facility provides advice on sequencing and sequence data analyzes, as well as on various aspects of planning and implementation of RT-PCR experiments.
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Sequencing Order Sheet
- Download | doc | 36 KB
Order for Genotyping
- Download | docx | 14 KB
PD Dr. rer. nat. Dipl.-Biol. Sabine Hoffmeister-Ullerich
ZMNH Core Facilty Bioanalytics
Rooms 1.21 and 1.22, 1st floor
Tel.: +49 (0) 40 7410 - 56659 and - 56662