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Analysis of Repetitive Regions in Myotonic Dystrophy Type 1 and 2
Publication Name:
Current Protocols in Human Genetics
Unit Number:
UNIT 9.6
DOI:
10.1002/0471142905.hg0906s61
Online Posting Date:
April, 2009 Abstract
Myotonic dystrophy is an autosomal dominant disorder characterized by myotonia, progressive muscle wasting, and cataracts. There are two forms identified: myotonic dystrophy type 1 (DM1), caused by an expansion of a CTG repeat in the 3¢ untranslated region of the myotonin-protein kinase (DMPK) gene on chromosome 19, and myotonic dystrophy type 2 (DM2), caused by an expansion of a CCTG repeat in intron 1 of the cellular nucleic acid–binding protein (CNBP) gene on chromosome 3. There is no single method that can identify all ranges of repeats in both disorders. Protocols in this unit describe the analysis of PCR-amplified CTG repeats from the DMPK gene and CCTG repeats from the CNBP gene, respectively, using a fluorescent-labeled primer followed by capillary electrophoresis. An additional protocol describes the analysis of genomic DNA by Southern blot and hybridization for DM1, while yet another describes a similar technique to analyze the repeat in DM2 using field-inversion gel electrophoresis. Both techniques identify 100% of cases of these two disorders. Curr. Protoc. Hum. Genet. 61:9.6.1-9.6.19. © 2009 by John Wiley & Sons, Inc.
Keywords: myotonic dystrophy type 1; myotonic dystrophy type 2; DMPK; CNBP
Table of Contents
- Introduction
- Basic Protocol 1: PCR Amplification of DMPK CTG Repeats
- Basic Protocol 2: PCR Amplification of CNBP Repeat
- Basic Protocol 3: Hybridization Analysis of Myotonic Dystrophy Type 1 (DM1) CTG Repeats in Genomic DNA
- Support Protocol 1: PCR Analysis of the Alu Insertion Polymorphism
- Basic Protocol 4: Hybridization Analysis of Myotonic Dystrophy Type 2 (DM2) CCTG Repeats in Genomic DNA
- Support Protocol 2: ZNF9 Probe Synthesis
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Other Versions
Materials
Basic Protocol 1: PCR Amplification of DMPK CTG Repeats
Materials
- 1.25 mM dNTP mix (appendix 2D)
- 10× PCR amplification buffer (appendix 2D)
- 50 mM MgCl
2 - 20 ng/µl primer 1 (5¢-CAG AGC AGG GCG TCA TGC ACA-3¢)
- 20 ng/µl primer 2 (FAM 5¢-GAA GGG TCC TTG TAG CCG GGA A-3¢) labeled with the fluorescent tag FAM (ABI)
- DMSO (molecular biology grade; Sigma)
- 1.5 µg/µl T4 gene protein 32 (Amersham)
- 5 U/µl Taq DNA polymerase
- 200 ng/ml DNA (minimum concentration) sample and positive control (known 70 to 80 repeats): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
- Deionized formamide (unit 4.3)
- Liz500 internal lane marker (ABI)
- POP-7 polymer (ABI)
- 0.2-ml PCR tubes
- Thermal cycler (e.g., ABI 9700)
- Capillary electrophoresis system (e.g., ABI 3130)
Basic Protocol 2: PCR Amplification of CNBP Repeat
Materials
- 1.25 mM dNTP mix (appendix 2D)
- 10× PCR amplification buffer (appendix 2D)
- 50 mM MgCl
2 - 20 ng/µl primer 1 (5¢-GCC TAG GGG ACA AAG TGA GA-3¢)
- 20 ng/µl primer 2 (FAM-5¢-GGC CTT ATA ACC ATG CAA ATG-3¢) labeled with the fluorescent tag FAM
- 5 U/µl Taq DNA polymerase
- 10% (w/v) Triton X-100 (Roche)
- 2 ng/ml DNA (minimum concentration) sample and positive controls (with two different repeat sizes): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
- Deionized formamide (unit 4.3)
- Liz500 internal lane marker (ABI)
- POP-7 polymer (ABI)
- 0.2-ml PCR tubes
- Thermal cycler (e.g., ABI 9700)
- Capillary electrophoresis system (e.g., ABI 3130)
Basic Protocol 3: Hybridization Analysis of Myotonic Dystrophy Type 1 (DM1) CTG Repeats in Genomic DNA
Materials
- 50 U/µl EcoRI restriction enzyme and 10× restriction endonuclease buffer
- Spermidine
- 3 to 4 µg per lane purified genomic DNA samples and controls (no CTG expansion and heterozygous for Alu, 80 repeats and a normal 10-kb allele, and an expanded allele with >1000 repeats): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
- 0.6% agarose gel (unit 2.7)
- 1× TBE buffer (appendix 2D)
- 10× gel loading buffer (appendix 2D)
- Molecular-size markers (e.g., -HindIII digested DNA, Invitrogen)
- 1 µg/ml ethidium bromide in 1× TBE buffer
- 0.4 M NaOH
- Neutralization solution: 0.2 M Tris·Cl, pH 7.5/2× SSC (see appendix 2D)
- Prehybridization/hybridization buffer (see recipe)
- pGB2.2 probe (available from the author)
- [-
32 P]dCTP (Perkin-Elmer) - Salmon sperm DNA (see recipe)
- Wash buffer: 0.1× SSC/1.0% SDS
- 0.5-ml microcentrifuge tubes
- 20 ×20–cm casting tray with 1.5-mm-thick comb
- Nylon membrane, positively charged (e.g., Biotrans+, ICN Biomedicals)
- 55°C to 65°C heating block
- Filter paper (Whatman 3 MM)
- Plastic wrap
- X-AR autoradiographic film with intensifying screen
- Additional reagents and equipment for preparing of genomic DNA (appendix 3B & unit 14.4), performing agarose gel electrophoresis and Southern blots (unit 2.7), and labeling DNA by random-hexamer priming and column-purifying (appendix 3E)
CAUTION: Radiolabeled probes are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
Support Protocol 1: PCR Analysis of the Alu Insertion Polymorphism
Materials
- 1.25 mM dNTP mix (appendix 2D)
- 10× PCR amplification buffer (appendix 2D)
- 50 mM MgCl
2 - 20 ng/µl primer 1 (5¢-AAA TAG GCT GCA CCG CGG-3¢)
- 20 ng/µl primer 2 (5¢-CTG TAT ACT CAG CTA CTA GGG T-3¢)
- 20 ng/µl primer 3 (5¢-CTC AGG GGT TAT CTA AAG TGG C-3¢)
- 5 U/µl Taq DNA polymerase
- 200 ng/ml DNA (minimum concentration) sample and positive controls (known Alu-plus and Alu-minus alleles): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
- 1% (w/v) agarose gel
- 10 mg/ml ethidium bromide
- 1× TBE
- 123-bp molecular size marker (e.g., Invitrogen)
- 0.2-ml PCR tubes
- Thermal cycler (e.g., ABI 9700)
- Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)
Basic Protocol 4: Hybridization Analysis of Myotonic Dystrophy Type 2 (DM2) CCTG Repeats in Genomic DNA
Materials
- 50 U/µl EcoRI restriction enzyme and 10× restriction endonuclease buffer
- 3 to 4 µg purified genomic DNA per lane (³50 kb fragments), sample and controls (no CCTG expansion and an expanded repeat): prepared using a salting out or column procedure (e.g., a Gentra kit; Invitrogen)
- Agarose
- 0.5× TBE buffer (see appendix 2D)
- 10× gel loading buffer
- Molecular-size markers (e.g., -HindIII digested DNA; Invitrogen)
- 10 µg/ml ethidium bromide in 0.5× TBE buffer
- 0.25 M HCl
- 0.4 M NaOH/1.5 M NaCl
- Neutralization solution: 0.2 M Tris·Cl (see appendix 2D), pH 7.5/2× SSC (see appendix 2D)
- Prehybridization/hybridization buffer (see recipe)
- ZNF9 probe (Support Protocol 2)
- [-
32 P]dCTP (Perkin-Elmer) - Human placental DNA (see recipe)
- Salmon sperm DNA (see recipe)
- Wash buffer: 0.1× SSC/0.1% SDS
- 0.5-ml microcentrifuge tubes
- Field-inversion gel-electrophoresis (FIGE) unit (e.g., Hoefer) or pulse-field gel-electrophoresis unit, and gel casting tray
- Nylon membrane, positively charged (e.g., Biotrans+, ICN Biomedicals)
- Filter paper (Whatman 3MM)
- 50°C and 65°C heating block
- X-AR autoradiographic film with intensifying screen
- Additional reagents and equipment for preparing genomic DNA (appendix 3B & unit 14.4), performing agarose gel electrophoresis and Southern blots (unit 2.7), and labeling DNA by random-hexamer priming and column-purifying (appendix 3E)
Support Protocol 2: ZNF9 Probe Synthesis
Materials
- 1.25 mM dNTP mix (appendix 2D)
- 10× PCR amplification buffer (appendix 2D)
- 50 mM MgCl
2 - 20 ng/µl primer 1 (5¢-GAG AAC CTT GCC ATT TTT CG-3¢)
- 20 ng/µl primer 2 (5¢-CAC CTA CAG CAC TGG CAA CA-3¢)
- 5 U/µl Taq DNA polymerase
- 10% (w/v) Triton X-100 (Roche)
- Cloned ZNF9 DNA (available by request from the author)
- 1% (w/v) agarose gel with ethidium bromide
- 0.2-ml PCR tubes
- Thermal cycler
- Transilluminator 1.5-ml microcentrifuge tubes
- Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)
Looking for materials? Suppliers Guide
Figures
-
Figure 9.6.1PCR amplification of the CTG repeat in the DMPK gene. PCR products were generated with a FAM fluorescent–tagged primer and electrophoresed using an ABI 3130 capillary electrophoresis instrument. Repeat sizes of 12 and 13 are indicated. * indicates stutter peaks. -
Figure 9.6.2PCR amplification of the CTG repeat in the DMPK gene. PCR products were generated with a FAM fluorescent–tagged primer and electrophoresed using an ABI 3130 capillary electrophoresis instrument. (A) Repeat sizes of 12 and an expansion of 84. (B) Close-up of the 84 repeat product, showing extensive stutter peaks. -
Figure 9.6.3PCR amplification of the CCTG repeat in the CNBP gene. PCR products were generated with a primer labeled with a FAM fluorescent tag and electrophoresed using an ABI 3130 capillary electrophoresis instrument (ABI). * indicates stutter peaks. (A) Repeat sizes of 140 bp and 142 bp are indicated. (B) Repeat sizes of 136 bp and 144 bp. -
Figure 9.6.4Structural organization of the DMPK region. E, EcoRI restriction enzyme site. The locations of the pGB2.2 probe, CTG repeat, and 1-kb Alu insert are indicated. Exons are depicted as boxes, and exons 1, 2, 9, and 15 are numbered. -
Figure 9.6.5Autoradiogram of hybridization analysis of genomic DNA hybridized with the pGB2.2 probe from the DMPK region. Normal 9- and 10-kb alleles are indicated. * indicates individuals with expanded CTG repeats. Lane 2 has a normal allele of 10-kb and an expanded CTG repeat of ~80 repeats, showing that electrophoresis has progressed far enough to separate these two alleles. Lane 3 has >1000 repeats and is visible as a smear. -
Figure 9.6.6Autoradiogram of hybridization analysis of genomic DNA hybridized with the pGB2.2probe from the DMPK region. Normal 9 and 10 kb alleles are indicated. * indicates individuals with expanded CTG repeats associated with the Alu-insert (10 kb) allele. ** indicates individuals with expanded CTG repeats associated with the Alu-minus (9 kb) allele. -
Figure 9.6.7Structural organization of the CNBP region. E, EcoRI restriction enzyme site. (A) The entire CNBP gene showing all 5 exons. (B) Close up of the EcoRI fragment. The locations of the ZNF9 probe and the CNBP repeat are indicated. -
Figure 9.6.8Autoradiogram of hybridization analysis of genomic DNA hybridized with the ZNF9 probe from the CNBP region. The normal 4.5-kb allele is indicated. * indicates individuals with expanded CCTG repeats. -
Figure 9.6.9Organization of the repeat unit in intron 1 of the CNBP gene. The range of repeats in normal alleles is given. Only the CCTG repeat is expanded in DM2.
Literature Cited
| Literature Cited | |
| Brook, J., McCurrach, M., Harley, H., Buckler, A., Church, D., Aburatani, H., Hunter, K., Stanton, V., Thirion, J-P., Hudson, T., Sohn, R., Zemelman, B., Snell, R.G., Rundle, S.A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Jones, C., Juvonen, V., Johnson, K., Harper, P.S., Shaw, D.J., and Housman, D. 1992. Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3¢ end of a transcript encoding a protein kinase family member. Cell 68:799-808. | |
| Day, J. W., Ricker, K., Jacobsen, J.F., Rasmussen, L.J., Dick, B.A., Kress, B.A., Schneider, C., Koch, M.C., Beilman, G.J., Harrison, A.R., Dalton, J.C., and Ranum, L.P.W. 2003. Myotonic Dystrophy Type 2: Molecular, diagnostic and clinical spectrum. Neurol. 60:657-664. | |
| Michael Finney, M. 2000. Pulsed-field gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5B.1-2.5B.9. | |
| Fu, Y.-H., Pizzuti, A., Fenwick, R.G., Jr., King, J., Rajnarayan, S., Dunne, P.W., Dubel, J., Nasser, G.A., Ashizawa, T., De Jong, P.J., Wieringa, B., Korneluk, R.G., Perryman, M.B., Epstein, H.F., and Caskey, C.T. 1992. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255:1256-1258. | |
| Harper, P.S. 1989. Myotonic Dystrophy, 2nd ed. W.B. Saunders, Philadelphia. | |
| Hunter, A., Tsilfidis, C., Mettler, G., Jacob, P., Mahadevan, M., Surh, L., and Korneluk, R. 1992. The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy. J. Med. Genet. 29:774-779. | |
| Jakubiczka, S., Vielhaber, S., Kress, W., Kupferling, P., Reuner, U., Kunath, B., and Wieacker, P. 2004. Improvement of the diagnostic procedure in proximal myotonic myopathy/myotonic dystrophy type 2. Neurogenet. 5:55-59. | |
| Krahe, R., Eckhart, M., Ogunniyi, A.O., Osuntokun, B.O., Siciliano, M.J., and Ashizawa, T. 1995. De novo myotonic dystrophy mutation in a Nigerian kindred. Am. J. Hum. Genet. 56:1067-1074. | |
| Liquori, C.L., Ricker, K., Moseley, M.L., Jacobsen, J.F., Kress, W., Naylor, S.L., Day, J.W., and Ranum, L.P.W. 2001. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864-867. | |
| Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K., Leblond, S., Earle-MacDonald, J., De Jong, P.J., Wieringa, B., and Korneluk, R. 1992. Myotonic dystrophy mutation: An unstable CTG repeat in the 3¢ untranslated region of the gene. Science 255:1253-1255. | |
| Ranum, L.P.W., Rasmussen, P.F., Benzow, K.A., Koob, M.D., and Day, J.W. 1998. Genetic mapping of a second myotonic dystrophy locus. Nat. Genet. 19:196-198. | |
| Second International Myotonic Dystrophy Consortium (IDMC). 2000. New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1). Neurol. 54:1218-1221. | |
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