Protocol
Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis

https://doi.org/10.1016/S1385-299X(96)00019-0Get rights and content

Abstract

Spontaneous neurologic mutations in the mouse provide powerful tools for the study of mammalian central nervous system development. The study of mouse neurologic mutants has led to a better understanding of the complex mechanisms involved in the development of the nervous system. Because few of these mutations have been identified, molecular probes distinguishing heterozygotes from homozygotes are generally unavailable. Further, most neurologic mouse mutants breed poorly as homozygotes, making it necessary to breed heterozygotes and select homozygous mutant progeny based on phenotype. The requirement for heterozygous breeding and the lack of molecular markers specific for the mutation have hampered developmental studies because the underlying neurologic perturbations occur before the mutant mice can be identified by phenotype. The recent identification and chromosomal assignment of simple sequence repeats (SSRs), repetitive sequences of DNA found at a high density throughout the mouse genome, provide the tools for mapping mutations in the mouse and for subsequent genotyping of potential mutants prior to phenotype onset. The SSRs are useful because these markers are polymorphic (for review see Weber, J.L., Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: K.E. Davies and S.M. Tilghman (Eds.), Genetic and Physical Mapping. Genome Analysis, Vol. 1, Cold Spring Harbor Laboratory Press, Plainview, NY, 1990, pp. 159–181 [16]), that is, the size of the individual SSRs differs among strains of mice. Following polymerase chain reaction (PCR) amplification of an SSR and separation of PCR products by polyacrylamide gel electrophoresis, one can easily visualize differences in the size of the PCR product between mouse strains. Many mutations in the mouse arose spontaneously on inbred strains and were subsequently backcrossed onto a different strain. After many generations of congenic backcrosses, the only DNA retained from the original mutant strain is composed of the mutant gene and closely linked regions. Thus, it is possible to cross the mutant strain to a different mouse strain and map the mutation by correlating mutant phenotype to SSRs the same size as the original mutant strain. We have mapped the tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr) mutations using SSRs in backcrossed mouse strains. The SSRs distinguishing mutant from normal strains can then be used to genotype potential mutant pups before the onset of the mutant phenotype. The protocol described below can be adapted to almost any mutation congenically inbred for genotyping. Here we describe a method for selecting primers appropriate for genotyping potential mouse mutants and a rapid protocol for genotype screening. Even with SSRs distinguishing mutant from normal mice, genotyping several mice simultaneously can be a daunting task. This is primarily because the protocols available for preparing DNA for PCR amplification are time-consuming, requiring several purification steps including phenol extractions. Although kits are commercially available for DNA preparation without organic extractions, these kits tend to be expensive. The protocol described is a rapid, inexpensive method of determining the genotype of mice using PCR analysis of dried blood spots. The protocol only requires PCR primers distinguishing among alleles and is therefore ideal for the rapid identification of potential mutants for those mouse mutations which have been mapped using microsatellite markers. The DNA preparation protocol may also be used in rapid screening of potential transgenic mice. © 1997 Elsevier Science B.V. All rights reserved.

Section snippets

Type of research

  • Identification of mouse mutants prior to phenotypic onset of genotype.

  • Identification of double mutant mice.

  • 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

  • 2.4 cm GF/C filters (Whatman International, Maidstone, UK).

  • Mouse ear-punch (Michi-Crown, Bay City, MI).

  • DNA Speed Vac (Savant Instruments, Farmingdale, NY).

  • UNO thermocycler (Biometra, Tampa, FL).

  • Sequencing gel apparatus (Jordan Scientific Co., Bloomington, IN).

  • Power supply.

  • 3M paper (Whatman International, Maidstone, UK).

  • Gel drier.

  • Vacuum pump (Savant Instruments, Farmingdale, NY).

  • X-ray film (Kodak X-OMAT AR, Rochester, NY).

  • Film developer.

Chemicals and reagents

  • Methanol (Sigma, St. Louis, MO).

  • 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]

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    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.

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