For Rapid Screening of the Entire Mitochondrial Genome using Multiplex DHPLCThe MitoScreen Assay Kit for the WAVE® System provides the reagents and protocols necessary to screen human genomic DNA populations for heteroplasmic mutations present in the mitochondrial genome. Denaturing high performance liquid chromatography (DHPLC) on the WAVE System has become a widely recognized technology for both efficient and highly sensitive discovery of known and unknown mutations1-3. Human genomic DNA is used as the starting material for amplification of the human mitochondrial genome using a total of 19 primer sets (Figure 1). The amplified overlapping fragments range in size from 300-1500bp. Four of these fragments are in the size range 300-550 bp and as such are used for conventional DHPLC analysis. The remaining 15 fragments undergo a restriction digestion step in order to produce a collection of fragments that are analyzed by multiplex DHPLC (Example given in Figure 2). The schematic in Figure 3 shows the 19 PCR fragments created and their location on the mitochondrial map. 
Figure 1: Schematic representation of the MitoScreen™ Assay Kit Protocol
Figure 2: Analysis of PCR fragment MT 10 in different pooled DNA samples at 55°C. A heteroplasmic polymorphism is clearly seen in the 5th fragment* of MT 10 in DNA sample 2 and 3 as indicated by the arrows. *Fragment 5 is a 504 bp fragment corresponding to nucleotides 7204-7707 of the mtDNA genome sequence.
Figure 3: Schematic Diagram of the Mitochondrial Genome with Amplicon Coverage indicated. Please note that the arrow position is only indicative, but that the exact range is given numerically.
Applications- Mutation screening for mitochondrial disorders, e.g. neuromuscular diseases, cognitive dysfunctions and diabetes
- Mutation screening of mitochondria in relation to cancer research
Features and Benefits| Contains all necessary reagent as well as a detailed protocol and a Method CD for screening the entire human mitochondrial Genome | No development work required, all kit components are readily available and fully compatible with each other and the WAVE System | | Protocol developed using Multiplex-DHPLC | Rapid, automated screening of the entire mitochondrial genome in less than 8 hours on a WAVE System 3500HT | | High Sensitivity of the WAVE Systems in combination with high fidelity Optimase® Polymerase | Allows the detection of low levels of heteroplasmic mutations (< 1% heteroplasmy for certain mutations1) | | All components are WAVE Optimized, quality controlled and produced in an ISO 9001 environment | Long lifetime for the DNASep® Cartridge, reduced cost and minimized instrument downtime |
Components of the MitoScreen Assay Kit|
| 707001 Order | | MitoScreen Assay Kit | | Reagents for 12 complete Mitochondrial scans | | 707002 Order | | MitoScreen Assay Kit Plus | | Reagents for 24 complete Mitochondrial scans |
Each kit contains the following: Primer Sets MT 1–19, Optimase® Polymerase, PCR reagents and control DNA, dNTPs and Restriction enzymes (see following table) as well as the MitoScreen Assay Protocol Booklet and the MitoScreen Assay Kit Method CD. The user will be able to carry out > 230 PCR reactions with the MitoScreen Assay Kit and > 460 PCR reactions with the MitoScreen Assay Kit Plus. Reagents for performing 12 and 24 complete Human Mitochondrial Genome Scans: |
| Mitochondrial Genome Amplification Primer Sets MT 1-19 (5 µM) | | 55 µl | | 110 µl | | Optimase® Polymerase 2.5 U/µl | | 400 µl | | 800 µl | | 10x PCR Reaction Buffer + MgSO4 | | 2000 µl | | 4000 µl | | 10mM Nucleotide Mix | | 1550 µl | | 3100 µl | | Nde II (5 U/µl) | | 66 µl | | 132 µl | | Hae III (10 U/µl) | | 80 µl | | 160 µl | | Dde I (10 U/µl) | | 47 µl | | 94 µl | | Msp I (10 U/µl) | | 47 µl | | 94 µl | | Alu I (10 U/µl) | | 66 µl | | 132 µl | | Control DNA (Human) | | 50 µl | | 50 µl |
ShippingThe MitoScreen Assay Kit is shipped on dry ice. Please store at -15 to -30°C. Minimum shelf life expected is 6 months after delivery if stored as directed. Quality ControlManufactured under ISO 9001 compliance. Quality Control of all individual components and the complete assay kit is performed. References- van den Bosch B.J.C., de Coo R.F.M., Scholte H.R., Nijland J.G., van den Bogaard R., de Visser M., de Die-Smulders C.E.M., Smeets H.J.M. (2000) Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography. Nucleic Acids Res., 28, e89. Abstract Library Entry
- Lui M.R., Pan K.F., Li Z.F., Wang Y., Deng D.J., Zhang L., Lu Y.Y. (2002) Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography. World J. Gastroenterol., 8, 426-430. Abstract Library Entry
- Jones A.C., Sampson J.R., Cheadle J.P. (2001) Low level mosaicism detectable by DHPLC but not by direct sequencing. Hum. Mut. 17, 233-234. Abstract Library Entry
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