ProtocolRapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis
Section snippets
Type of research
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Identification of mouse mutants prior to phenotypic onset of genotype.
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Identification of double mutant mice.
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Transgenic mouse screening.
Time required
The entire protocol can be completed in 2 days, one of those days being blood collection and drying of the blood spots. Preparation of DNA for PCR analysis takes only a few minutes of hands-on time.
Collection of blood from each mouse requires about 15 s. The blood spots are dried overnight. The preparation of blood for PCR analysis takes about 25 min. End-labeling of the PCR primer requires 65 min. The polymerase chain reaction takes 2.5–3 h. The addition of formamide and denaturation of the
Special equipment
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2.4 cm GF/C filters (Whatman International, Maidstone, UK).
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Mouse ear-punch (Michi-Crown, Bay City, MI).
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DNA Speed Vac (Savant Instruments, Farmingdale, NY).
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UNO thermocycler (Biometra, Tampa, FL).
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Sequencing gel apparatus (Jordan Scientific Co., Bloomington, IN).
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Power supply.
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3M paper (Whatman International, Maidstone, UK).
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Gel drier.
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Vacuum pump (Savant Instruments, Farmingdale, NY).
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X-ray film (Kodak X-OMAT AR, Rochester, NY).
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Film developer.
Chemicals and reagents
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Methanol (Sigma, St. Louis, MO).
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T4 polynucleotide kinase
Detailed procedure
This section describes the procedure for genotyping potential mutant animals. Because several protocols are available for PCR and for polyacrylamide gel electrophoresis, we briefly describe the protocol we use. In contrast, the identification of PCR primers and preparation of DNA for PCR analysis is described in detail.
A. Identify PCR primers distinguishing mutant from normal alleles
This procedure can be approached from three starting points: screening potential transgenic mice, screening mice
Results
The results will consist of a single band or two bands per lane. A single band identifies the mouse as a homozygote. Two bands in one lane identify the mouse as a heterozygote. The size of the bands will depend on the PCR primers used. For example, we routinely genotype progeny from heterozygous tottering (C57BL/6J −+/tg) breeder pairs with simple sequence repeat D8Mit104. PCR amplification of tottering (tg/tg) mutant mouse DNA produces a single band at 139 base pairs (bp) corresponding to the
Discussion
The strength of this protocol is the rapid, inexpensive, nearly labor-free isolation of genomic DNA for PCR analysis from very small volumes of blood and the ability to identify mouse mutants early in life. Many mouse mutations, including tottering (tg) [1], Purkinje cell degeneration (pcd) [1], nervous (nr) [1], lurcher (Lc) [18], weaver (wv) [13], and reeler (rl) [14]have been localized to precise regions of the mouse genome using simple sequence length polymorphisms 3, 4, 5, 17. For each of
Quick procedure
A. Identify PCR primers distinguishing mutant from normal alleles.
B. Using a razor blade or scalpel, nick the tail vein.
C. Spot blood samples on Whatman GF/C filter paper.
D. Allow the blood samples to dry overnight.
E. Clean a standard mouse ear-punch by rinsing in 2.5 N HCl followed by distilled water.
F. Punch several holes in a clean piece of filter paper.
G. Punch a single 2 mm disk of blood sample into a clean Eppendorf tube.
H. Add 10 μl methanol and allow to incubate 15 min.
I. Remove
Essential literature references
Original papers: [1], [4], [8]
References (18)
- Campbell, D.B. and Hess, E.J., Chromosomal localization of the neurological mouse mutations tottering (tg), Purkinje...
- Ceci, J.D., Mouse chromosome 8, Mammalian Genome, 5, Spec. (1994)...
- Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Nadeau, J.H., Eppig, J.T., Maltais, L.J., Miller, J.C., Dietrich, W.F.,...
- Dietrich, W.F., Miller, J., Steen, R., Merchant, M.A., Damron-Boles, D., Husain, Z., Dredge, R., Daly, M.J., Ingalls,...
- Dietrich, W.F., Miller, J.C., Steen, R.G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D.C., Wessel, M.,...
- Doolittle, D.P., Davisson, M.T., Guidi, J.N. and Green, M.C., Catalog of mutant genes and polymorphic loci. In: M.F....
- Ghetti, B., Triarhou, L.C., Alyea, C.J., Dlouhy, S.R. and Karn, R.C., Unique cerebellar phenotype combining granule and...
- Gregory, C.A., Myal, Y. and Shiu, R.P.C., Rapid genotyping of transgenic mice using dried blood spots from Guthrie...
- Landis, S.C., Changes in neuronal mitochondrial shape in brains of nervous mutant mice, J. Hered., 64 (1973)...
Cited by (15)
Genotyping of transgenic mice: Old principles and recent developments
2005, Analytical BiochemistryCitation Excerpt :Although it is much more labor intensive, this technique can be useful for determination of zygosity. A great advantage of using blood spots [55,56] is that samples can be easily stored over a long period of time, enabling the analysis to be carried out at one’s convenience. On the other hand, the use of hairs [57–59], oral material [60,61], rectum cells [62], or even stool [63] as a source of DNA represents a great improvement in animal welfare because suffering is reduced significantly.
Tottering mouse motor dysfunction is abolished on the Purkinje cell degeneration (pcd) mutant background
1999, Experimental NeurologyN-glycosylation analysis of mouse immunoglobulin G isolated from dried blood spots
2021, ElectrophoresisAdverse effects of vapocoolant and topical anesthesia for tail biopsy of preweanling mice
2015, Journal of the American Association for Laboratory Animal Science