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Chapter 11 Gene Mapping in Zebrafish Using Single-Strand Conformation Polymorphism Analysis
CHAPTER 11
Gene Mapping in Zebrafish Using Single-Strand Conformation Polymorphsm Analysis Dorothee Foernzler and David R. Beier’ Genetics Division Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts 021 15
I. Introduction 11. SSCP Methodology 111. Gene Mapping IV. Methods A. Primer Labeling B. PCR Reaction C. SSCP Gel Electrophoresis References
I. Introduction Positional cloning or a candidate gene analysis will be necessary to characterize the large number of genes causing developmental abnormalities in zebrafish generated by genome-wide mutagenesis screens (Haffter et ab, 1996; Driever et al., 1996). A prerequisite for both of these strategies is a high-resolution genetic map of the zebrafish genome. Even though maps constructed using random amplified polymorphic DNAs (RAPDs) ( Johnson et al., 1996) and microsatellites (Knapik et al., 1996) will facilitate positional cloning efforts, the success of a candidate gene approach will depend on the efficient localization of expressed Corresponding author: Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, 20 Shattuck Street, Boston, Massachusetts 02115 METHODS IN CELL BIOLOGY,VOL. 60 Copynght 0 1999 by Acidenuc Prer, All ngha ofreproducoon m my form reserved. CG91-679X199 130.00
185
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Dorothee Foernzler and David R. Beier
sequences. In this chapter, we will introduce single-strand conformation polymorphism (SSCP) analysis as a powerful tool to map cDNAs in the zebrafish. Originally developed for the detection of single-base-pair mutations at specific loci (Orita et af., 1989), SSCP takes advantage of the fact that even singlenucleotide changes in short fragments of single-strand DNA may alter the intramolecular secondary conformation of these DNA stretches. If a short DNA fragment is denatured at high temperature and then quickly cooled, renaturation into double-stranded DNA is inhibited, and each single strand will assume its most-favored conformation according to its lowest free-energy state. This conformation is affected by changes in nucleotide content, which, under appropriate conditions, will often detectably shift its migration in a nondenaturing electrophoresis gel. Variant alleles identified by different migration patterns represent single-strand conformation polymorphisms. Linkage studies using SSCP analyses have been successfully applied in the mouse using recombinant inbred strains or interspecific crosses (Beier et af., 1992; Beier, 1993; Brady et af., 1997). This technique has been extended for mapping genes in several other organisms including humans. It has been shown to be simple, rapid, and reproducible. No special equipment not common to any molecular biology laboratory is required. The PCR-based approach requires only small amounts of DNA, which makes it especially attractive for organisms for which the amount of DNA recoverable per animal is limited, as is the case for zebrafish. The polymorphisms detected using this technique are stably inherited, and SSCP can be applied to map any unique DNA region in a cross of sufficiently divergent strains. A disadvantage of this mapping strategy, in contrast to RFLP analysis by hybridization, is the requirement of species-specific sequence. Another limitation for the use of SSCP in linkage studies is the necessity for a sufficient polymorphism frequency. This, of course, is not specific for this form of analysis but is true for any sequence-dependent mapping technique. SSCP applied for the purpose of gene mapping requires the PCR-mediated amplification of short DNA fragments derived from genomic sequences. Sequence templates for primer design ideally will correspond to regions less likely to be conserved to increase the chance of a polymorphic PCR product. For this purpose, 3’ untranslated regions and introns serve very well, although information regarding the latter is usually unavailable for cDNAs. To assess the polymorphism frequency of expressed sequences in commonly used zebrafish strains, we have tested over 150 primer pairs derived from 3’UTR. Every primer pair was initially tested for the presence of a polymorphism in the 3’UTR in parental DNA before mapping analyses with a reference cross DNA panel was performed. A maximal polymorphism frequency of 31% was detected for the AB and India strains used by Knapik et al., (1996), and 42% for the SJD and C32 strains used by Johnson er af. (1996) (Foernzler et af., 1998, submitted). As a comparison, the maximal polymorphism frequency in 3’ UTR sequences between inbred strains of mice was 20%, and in the two different species Mus musculus and Mus spretus, it was 70% (Brady et af., 1997). The zebrafish lines used have been inbred for several
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generations or are clonal derivatives of a presumptive ancestor, but they cannot be considered as completely inbred. The number of genetically fixed loci in the genomes used for linkage analysis will influence the complexity of SSCP patterns. We determined the strain distribution pattern of 17 polymorphic loci in zebrafish mapping crosses. On the MGH Reference Cross corresponding to the SSLP map (Knapik el al., 1996), 6 loci polymorphic between AB/India were mapped, and 3 loci polymorphic between C32 and SJD were localized on the Oregon mapping cross (Johnson et al., 1996). An example of this analysis is shown in Fig. 1. We included in our polymorphism screen 36 primers derived from partially characterized cDNA clones from kidney and embryonic cDNA libraries. In 13 of these expressed sequence tags, (ESTs) polymorphisms were detected, and 2 have been mapped. This demonstrates the feasibility of using ESTs for gene mapping in the zebrafish, which could be employed to generate a dense transcript map.
11. SSCP Methodology For SSCP analysis, a polymerase chain reaction (PCR) is performed to amplify fragments between 150 and 350 bp. For best results, the template DNA should be diluted to a DNA concentration of 50 ng, although we successfully amplified PCR products with as little as 16 ng of zebrafish DNA per reaction on a regular basis. Excess template DNA will result in decreased SSCP sensitivity. The choice of primers is often critical to the success of SSCP linkage studies and should be done carefully. To design PCR primers for SSCP analysis, at least
Fig. 1 SSCP analysis of expressed sequences. Genotype distributions of two different primer pairs corresponding to 3'UTR of expressed sequences are shown in progeny of two reference crosses. Top: Reference cross described by Knapik et al. (19%). Lanes correspond to diploid progeny of AB X India intercross. A: AB homozygote; H: heterozygote; I: India homozygote. Bottom: Reference cross described by Johnson et al. (1996). Lanes correspond to haploid progeny of SJD X C32 intercross. S: SJD; C C32.
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150 bp of unique sequence from less conserved regions such as 3’UTR, S’UTR, or intron must be available. These sequences should be screened for possible vector contaminants, cloning artifacts, or repetitive regions using similarity search programs such as BLAST. A number of computer programs for optimized design of PCR primers are available. For our analyses, PCR primers were designed from 3’UTR of cDNAs using the Primer 0.5 program (Lincoln er al., 1991) and the following criterion: primer length between 18 and 22 bp, primer GC% between 20 and 8096, primer melting temperature between 53°C and 60°C. A revised version of this program is Primer 3.0, which is available at http:// www.genome.wi.mit.edu/genomesoftware/other/primer3.html. Primers are 5’-end-labeled with f”P using T4-polynucleotide kinase, which we have found to be more efficient and more sensitive than labeling using incorporation of radioactive nucleotides during the PCR reaction. Even though it is usually sufficient to label only one of the PCR primers, testing both primers is more efficient for polymorphism detection because, in some cases, the mobility of only one strand will be affected by a sequence change. Most of the PCR primers designed using the criteria previously described will work under the standard PCR conditions that follow, but in some cases PCR conditions have to be optimized. If a primer pair does not amplify a product with the standard protocol, changing the Mg2’ concentration (within a range of 1.5-3 mM final concentration) or lowering the annealing temperature may result in more successful amplification.In contrast, an increase of the annealing temperature results in more specific PCR amplification in cases where the pattern complexity precludes unambigous scoring. To obtain single-strand DNA, the fragments are denatured and electrophoresed on a nondenaturing polyacrylamide gel at 4°C. Standardized electrophoresis conditions are highly recommended and require special attention in order to ensure reproducible results because the pattern of single-strand migration is very sensitive to a number of different parameters including gel matrix composition, buffer concentration, and temperature. We use a 5% polyacrylamide gel matrix for the separation of the single-strand fragments. The SSCP electrophoresis runs for 2 hours at 40 W in a 4°C cold room. It is crucial to keep the temperature low because heating the gel will denature the intrastrand secondary structure that generates the polymorphic pattern of migration. If a means to cool the gel is unavailable, it can be run at low power for longer times (e.g., overnight). The gel is then transferred to a filter paper, dried, and autoradiographed overnight. Amplifying screens are nor employed because they will result in diffuse bands and will decrease resolution. If maximal sensitivity is required (e.g., for very subtle polymorphisms), we have had success using a matrix sold by FMC BioProducts called MDE (Mutation Detection Enhancement) that is designed to enhance the separation of single-stranded products (Soto and Sukumar, 1992). However, the relative expense (compared with acrylamide gels) precludes its routine use in our lab. Additionally, the MDE matrix requires careful optimiza-
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tion of electrophoresis conditions because the results obtained are sensitive to even slight changes in the protocol. A variety of strategies have been suggested for increasing the efficiency of detecting SSCPs, including varying the TBE buffer concentration, varying the concentration of cross-linker in the gel matrix, adding glycerol to the gel matrix, and other modifications. In our own experience, we have found that the most important variable for polyacrylamide gel analysis run in the cold is the buffer concentration, which should be 0.5X TBE. If increased resolution is required, analysis using MDE matrix has generally proven satisfactory.
111. Gene Mapping This discussion of the utility of SSCP analysis for gene mapping in zebrafish presumes the availability of DNA from a mapping panel in which a sufficient number of markers have been typed such that assignment of a map position by linkage analysis is feasible. The general strategy for creating panels of this sort are illustrated by Johnson et al. (1996) and Knapik et al. (1996). The generation of diploid intercross or backcross progeny from a cross of divergent zebrafish strains is straightforward. The generation of mapping panels from haploid embryos is more complex; however, these data sets do not have heterozygous progeny, and the assignment of genotypes can, in many instances, be more conclusively scored (see Fig. 1). Before a primer pair is selected for SSCP mapping analysis, it is tested for a polymorphism in parental DNA of the strains used to generate the mapping panel. It is often necessary to test several primer pairs (which ideally are nonoverlapping) to find one with an easily resolved polymorphism. If a scorable polymorphism is found, the mapping panel is tested, and the allele distribution for the gene locus is determined. The Mapmanager software (Manly, 1993; available at http//:mcbio.med.buffalo.edu/mapmgr.html) is highly recommended for recording genotype distribution data and for determining linkage. Originally developed for genetic mapping in the mouse, it is useful for the analysis of genetic mapping experiments using backcrosses, intercrosses, recombinant inbred (RI) strains, or radiation hybrids. It facilitates the storage, retrieval, and display of genotype data and includes tools for statistical analysis of the experimental results. Of particular utility is a simple interface for positioning new genes on a reference cross. There are a number of potential difficulties in this procedure about which the investigator should be aware. In theory, a polymorphic fragment can be found in any expressed sequence as long as sufficient noncoding sequence is available. The efficiency of finding a polymorphic allele depends, to a considerable degree, on the size of the screened DNA region. If the 3’UTR is large enough, multiple primer pairs may be designed (which should ideally test minimally overlapping
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sequences) and tested for polymorphisms,or the use of intron or 5’UTR sequence can be employed for primer design. Discerning whether an apparent SSCP is “real” can require some experience. It is not uncommon that minor bands will be variably detectable, and their presence may be incorrectly assessed as being strain-specific. A simple means to reduce this source of error is to perform duplicate reactions using the parental DNAs of the mapping cross and to load these samples in alternating sequence. A true strain-specific shift can generally be readily recognized. Complexity of the SSCP patterns may cause problems. In some cases, multiple independently segregating products are found for one primer pair. Again, testing several PCR primer combinations derived from a sequence of interest may clarify which polymorphism corresponds to the locus. Alternatively, it is possible to elute a band identified as polymorphic, reamplify the fragment, and determine its sequence. In summary, SSCP mapping is an efficient tool to localize transcribed sequences in the zebrafish genome. Moreover, SSCP mapping is not restricted to fully characterized genes but can be used for localization of ESTs, similar to the efforts in human (Adams et al., 1991) and mouse (Brady et al., 1997). Because SSCP analysis can be used to map any unique genomic sequence, it is furthermore useful for analyzing sequences derived from the ends of large-insert clones, such as BACs, PACs, or YACs. For example, to assess possible chimerism in these clones, one can test that both ends colocalize. Additionally, in a chromosomal walk to a establish a contig over a region of interest, SSCP analyses may be helpful to determine the orientation of large-insert clones.
IV. Methods A. Primer Labeling 1. Sufficientsignal strength can obtained by labeling only one of the oligonucleotide primers, although maximal polymorphism detection is facilitated by labeling both. One microliter per reaction of a labeled primer mix prepared as follows will be used. The example is sufficient for 30 reactions: 24.2 p1 3.0 p1 1.0 p1 1.0 pl 1.2 p1 0.6 pl
dH2O 1OX T4 polynucleotide kinase buffer2 20pM forward primer 20pM reverse primer Y2P dATP, 6000Ci/mmole, 10mCilpl T4 polynucleotide kinase (10 ulpl)
2. Incubate at 37°C for 35-45 minutes. 1OX polynucleotide kinase buffer: 0.5M Tris-HCI pH 7.6; 0.1M MgC12; 0.05M Dithiothreitol; 1mM Spermidine-HCI; 1mM EDTA pH 8.0.
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3. Heat inactivate the reaction at 68°C for lominutes. 4. Keep reaction on ice until needed or store at -20°C. B. PCR Reaction
1. All PCR reactions are carried out in 96-well plates with a final reaction volume of 12.5 p1, and 1pl of template DNA (16-50 ng) is added to each well. For mapping panels that are to be analyzed for many markers, it is convenient to aliquot template DNA into plates and allow it to air-dry. These plates can then be stored. The dried DNA is readily rehydrated during PCR. 2. Use 9.25 p1 per reaction of a PCR master mix prepared as follows. The example is sufficient for 30 reactions: 205 p1 37.5 pl 2.5 pl 2.5 pl 30 pl
dH2O 1OX PCR buffer3 20pM forward primer 20p.M reverse primer ~ ~ ~ P - l a b eprimer l e d mix
3. Add 9.25 pl of the PCR master mix to each DNA template. 4. Mix well and overlay with 30 pl light mineral oil. 5. Mix on ice 0.5 p1 of 10mM each dNTP (dATP, dCTP, dGTP, dTTP) and 1 unit of Taq polymerase for each PCR. 6. Perform hot-start PCR: Denature the PCR reaction at 94°C for 5 minutes; hold at 80°C. 7. Add 2.25 p1 of dNTP/Taq mix to each reaction; mix well. 8. Amplify using appropriate thermocycling conditions for the primers used in the reaction. Our standard PCR conditions follows:
29-40 cycles: annealing: extension: denaturing: final cycle: annealing: extension:
55°C for 2 minutes 72°C for 3 minutes 94°C for 1 minutes 55°C for 2 minutes 72°C for 7 minutes
9. Store PCR at -20°C or keep on ice until needed. C. SSCP Gel Electrophoresis
1. Prepare gel mix for 5% polyacrylamide SSCP gel. Prepare 80 ml (sufficient for the Gibco BRL S2 sequencing gel apparatus used with a 0.4-mm spacer) as follows: 1OX PCR buffer: 0.1M Tris-HC1 pH
8.0;0.5M KCI; 0.02M MgC12; 0.01% gelatin.
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66 ml 4 ml 10 ml 800 PI 30pl
dHzO 1OX TBE (final concentration = OSX) acrylamide mix (38% acrylamide:2% Bis-acrylamide) 10% APS (ammoniumperoxodisulfate) in HzO, freshly prepared TEMED
2. Pour SSCP gel, insert comb, clamp well, and allow to polymerize for 2 hours or overnight. Avoid any bubbles in the gel. 3. Pre-equilibrate polymerized gel in gel apparatus with 0.5X TBE for at least 30 minutes at 4°C. 4. For each reaction, mix 2 p1 PCR product and 4 p1 USB stop solution solution (95% formamide; 0.05% bromphenol blue; 0.05% xylene cyanol; 0.02M EDTA pH 8.0) in a 96-well plate. Denature at 94°C for 5 minutes, transfer immediately onto ice. 5. Load 3-5 p1 per reaction onto gel. Run the gel for 90-150 minutes (according to the size for PCR product) in 0.5X TBE at 4°C and 40 W with constant power. 6. Transfer the gel onto Whatman filter paper, cover with plastic wrap, and dry on a gel dryer for 30 minutes. 7. Expose the gel to X-ray film overnight; do not use amplifying screens. References Adams, M., Kelley, J., Gocayne, J., Dubnick, M., Polymeropoulos, M., Xiao, H., Merril, C., Wu, A., Olde, B. and Moreno, R. (1991). Complementary DNA sequencing: Expressed sequence tags and human genome project. Science 252, 1651-1656. Beier, D. R., Dushkin, H. and Sussman, D. J. (1992). Mapping genes in the mouse using singlestrand conformation polymorphismanalysis of recombinant inbred strains and interspecificcrosses. Proc. Natl. Acad. Sci. U.S.A. 89, 9102-9106. Beier, D. R. (1993). Single-strand conformation polymorphism (SSCP) analysis as a tool for genetic mapping. Mamm. Genome 4,627-631. Brady, K. P., Rowe, L. B., Her, H., Stevens, T. J., Eppig, J., Sussman, D. J., Sikela, J., and Beier, D. R. 1997. Genetic mapping of 262 loci derived from expressed sequences in a murine interspecific cross using single-strand conformational polymorphism analysis. Genome Res. 7, 1085-1093. Driever, W., Solnica-Krezel,L., Schier, A. F., Neuhauss, S. C. F., Malicki, J., Stemple, D. L., Stainier, D. Y. R., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J. and Boggs, C. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123,37-46. Foernzler, D., Her, H., Knapik, E. W., Clark, M., Lehrach, H., Posthlethwait, J. H., Zon, L. I., and Beier, D. R. (1998). Gene mapping in zebrafish using single-strand conformation polymorphism analysis. Genomics, in press. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eden, F. J. M., Jiang, Y.-J., Heisenberg, C.-P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C. and Niisslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36. Johnson, S. L., Gates, M. A., Johnson, M., Talbot, W. S., Home, S., Baik, K., Rude, S., Wong, J. R., and Posthlethwait,J. H. (1996). Centromere-linkage analysis and consolidation of the zebrafish genetic map. Genetics 142,1277-1288.
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Knapik, E. W., Goodman, A., Atkinson, 0. S., Roberts, C. T., Shiozawa, M., Sim, C. U., WekslerZangen, S., Trolliet, M. R., Futrell, C., Innes, B. A,, Koike, G., McLaughlin, M. G., Pierre, L., Simon, J. S., Vilallonga, E., Roy, M., Chiang, P.-W., Fishman, M. C., Driever, W., and Jacob, H. J. (1996). A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development 123,451-460. Lincoln, S. E., Daly, M. J., and Lander, E. S. (1991). PRIMER: A computer program for automatically selecting PCR primers. Available from [email protected]. Manly, K. F. (1993). A Macintosh program for storage and analysis of experimental genetic mapping data. Mamm. Genome 4,303-313. Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5,74-879. Soto, D., and Sukumar, S. (1992). Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA. PCR Methods Applic. 2, 96-98.
Gene Mapping in Zebrafish Using Single-Strand Conformation Polymorphsm Analysis Dorothee Foernzler and David R. Beier’ Genetics Division Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts 021 15
I. Introduction 11. SSCP Methodology 111. Gene Mapping IV. Methods A. Primer Labeling B. PCR Reaction C. SSCP Gel Electrophoresis References
I. Introduction Positional cloning or a candidate gene analysis will be necessary to characterize the large number of genes causing developmental abnormalities in zebrafish generated by genome-wide mutagenesis screens (Haffter et ab, 1996; Driever et al., 1996). A prerequisite for both of these strategies is a high-resolution genetic map of the zebrafish genome. Even though maps constructed using random amplified polymorphic DNAs (RAPDs) ( Johnson et al., 1996) and microsatellites (Knapik et al., 1996) will facilitate positional cloning efforts, the success of a candidate gene approach will depend on the efficient localization of expressed Corresponding author: Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, 20 Shattuck Street, Boston, Massachusetts 02115 METHODS IN CELL BIOLOGY,VOL. 60 Copynght 0 1999 by Acidenuc Prer, All ngha ofreproducoon m my form reserved. CG91-679X199 130.00
185
186
Dorothee Foernzler and David R. Beier
sequences. In this chapter, we will introduce single-strand conformation polymorphism (SSCP) analysis as a powerful tool to map cDNAs in the zebrafish. Originally developed for the detection of single-base-pair mutations at specific loci (Orita et af., 1989), SSCP takes advantage of the fact that even singlenucleotide changes in short fragments of single-strand DNA may alter the intramolecular secondary conformation of these DNA stretches. If a short DNA fragment is denatured at high temperature and then quickly cooled, renaturation into double-stranded DNA is inhibited, and each single strand will assume its most-favored conformation according to its lowest free-energy state. This conformation is affected by changes in nucleotide content, which, under appropriate conditions, will often detectably shift its migration in a nondenaturing electrophoresis gel. Variant alleles identified by different migration patterns represent single-strand conformation polymorphisms. Linkage studies using SSCP analyses have been successfully applied in the mouse using recombinant inbred strains or interspecific crosses (Beier et af., 1992; Beier, 1993; Brady et af., 1997). This technique has been extended for mapping genes in several other organisms including humans. It has been shown to be simple, rapid, and reproducible. No special equipment not common to any molecular biology laboratory is required. The PCR-based approach requires only small amounts of DNA, which makes it especially attractive for organisms for which the amount of DNA recoverable per animal is limited, as is the case for zebrafish. The polymorphisms detected using this technique are stably inherited, and SSCP can be applied to map any unique DNA region in a cross of sufficiently divergent strains. A disadvantage of this mapping strategy, in contrast to RFLP analysis by hybridization, is the requirement of species-specific sequence. Another limitation for the use of SSCP in linkage studies is the necessity for a sufficient polymorphism frequency. This, of course, is not specific for this form of analysis but is true for any sequence-dependent mapping technique. SSCP applied for the purpose of gene mapping requires the PCR-mediated amplification of short DNA fragments derived from genomic sequences. Sequence templates for primer design ideally will correspond to regions less likely to be conserved to increase the chance of a polymorphic PCR product. For this purpose, 3’ untranslated regions and introns serve very well, although information regarding the latter is usually unavailable for cDNAs. To assess the polymorphism frequency of expressed sequences in commonly used zebrafish strains, we have tested over 150 primer pairs derived from 3’UTR. Every primer pair was initially tested for the presence of a polymorphism in the 3’UTR in parental DNA before mapping analyses with a reference cross DNA panel was performed. A maximal polymorphism frequency of 31% was detected for the AB and India strains used by Knapik et al., (1996), and 42% for the SJD and C32 strains used by Johnson er af. (1996) (Foernzler et af., 1998, submitted). As a comparison, the maximal polymorphism frequency in 3’ UTR sequences between inbred strains of mice was 20%, and in the two different species Mus musculus and Mus spretus, it was 70% (Brady et af., 1997). The zebrafish lines used have been inbred for several
11. Gene Mapping in Zebrafish Using SSCP Analysis
187
generations or are clonal derivatives of a presumptive ancestor, but they cannot be considered as completely inbred. The number of genetically fixed loci in the genomes used for linkage analysis will influence the complexity of SSCP patterns. We determined the strain distribution pattern of 17 polymorphic loci in zebrafish mapping crosses. On the MGH Reference Cross corresponding to the SSLP map (Knapik el al., 1996), 6 loci polymorphic between AB/India were mapped, and 3 loci polymorphic between C32 and SJD were localized on the Oregon mapping cross (Johnson et al., 1996). An example of this analysis is shown in Fig. 1. We included in our polymorphism screen 36 primers derived from partially characterized cDNA clones from kidney and embryonic cDNA libraries. In 13 of these expressed sequence tags, (ESTs) polymorphisms were detected, and 2 have been mapped. This demonstrates the feasibility of using ESTs for gene mapping in the zebrafish, which could be employed to generate a dense transcript map.
11. SSCP Methodology For SSCP analysis, a polymerase chain reaction (PCR) is performed to amplify fragments between 150 and 350 bp. For best results, the template DNA should be diluted to a DNA concentration of 50 ng, although we successfully amplified PCR products with as little as 16 ng of zebrafish DNA per reaction on a regular basis. Excess template DNA will result in decreased SSCP sensitivity. The choice of primers is often critical to the success of SSCP linkage studies and should be done carefully. To design PCR primers for SSCP analysis, at least
Fig. 1 SSCP analysis of expressed sequences. Genotype distributions of two different primer pairs corresponding to 3'UTR of expressed sequences are shown in progeny of two reference crosses. Top: Reference cross described by Knapik et al. (19%). Lanes correspond to diploid progeny of AB X India intercross. A: AB homozygote; H: heterozygote; I: India homozygote. Bottom: Reference cross described by Johnson et al. (1996). Lanes correspond to haploid progeny of SJD X C32 intercross. S: SJD; C C32.
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150 bp of unique sequence from less conserved regions such as 3’UTR, S’UTR, or intron must be available. These sequences should be screened for possible vector contaminants, cloning artifacts, or repetitive regions using similarity search programs such as BLAST. A number of computer programs for optimized design of PCR primers are available. For our analyses, PCR primers were designed from 3’UTR of cDNAs using the Primer 0.5 program (Lincoln er al., 1991) and the following criterion: primer length between 18 and 22 bp, primer GC% between 20 and 8096, primer melting temperature between 53°C and 60°C. A revised version of this program is Primer 3.0, which is available at http:// www.genome.wi.mit.edu/genomesoftware/other/primer3.html. Primers are 5’-end-labeled with f”P using T4-polynucleotide kinase, which we have found to be more efficient and more sensitive than labeling using incorporation of radioactive nucleotides during the PCR reaction. Even though it is usually sufficient to label only one of the PCR primers, testing both primers is more efficient for polymorphism detection because, in some cases, the mobility of only one strand will be affected by a sequence change. Most of the PCR primers designed using the criteria previously described will work under the standard PCR conditions that follow, but in some cases PCR conditions have to be optimized. If a primer pair does not amplify a product with the standard protocol, changing the Mg2’ concentration (within a range of 1.5-3 mM final concentration) or lowering the annealing temperature may result in more successful amplification.In contrast, an increase of the annealing temperature results in more specific PCR amplification in cases where the pattern complexity precludes unambigous scoring. To obtain single-strand DNA, the fragments are denatured and electrophoresed on a nondenaturing polyacrylamide gel at 4°C. Standardized electrophoresis conditions are highly recommended and require special attention in order to ensure reproducible results because the pattern of single-strand migration is very sensitive to a number of different parameters including gel matrix composition, buffer concentration, and temperature. We use a 5% polyacrylamide gel matrix for the separation of the single-strand fragments. The SSCP electrophoresis runs for 2 hours at 40 W in a 4°C cold room. It is crucial to keep the temperature low because heating the gel will denature the intrastrand secondary structure that generates the polymorphic pattern of migration. If a means to cool the gel is unavailable, it can be run at low power for longer times (e.g., overnight). The gel is then transferred to a filter paper, dried, and autoradiographed overnight. Amplifying screens are nor employed because they will result in diffuse bands and will decrease resolution. If maximal sensitivity is required (e.g., for very subtle polymorphisms), we have had success using a matrix sold by FMC BioProducts called MDE (Mutation Detection Enhancement) that is designed to enhance the separation of single-stranded products (Soto and Sukumar, 1992). However, the relative expense (compared with acrylamide gels) precludes its routine use in our lab. Additionally, the MDE matrix requires careful optimiza-
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tion of electrophoresis conditions because the results obtained are sensitive to even slight changes in the protocol. A variety of strategies have been suggested for increasing the efficiency of detecting SSCPs, including varying the TBE buffer concentration, varying the concentration of cross-linker in the gel matrix, adding glycerol to the gel matrix, and other modifications. In our own experience, we have found that the most important variable for polyacrylamide gel analysis run in the cold is the buffer concentration, which should be 0.5X TBE. If increased resolution is required, analysis using MDE matrix has generally proven satisfactory.
111. Gene Mapping This discussion of the utility of SSCP analysis for gene mapping in zebrafish presumes the availability of DNA from a mapping panel in which a sufficient number of markers have been typed such that assignment of a map position by linkage analysis is feasible. The general strategy for creating panels of this sort are illustrated by Johnson et al. (1996) and Knapik et al. (1996). The generation of diploid intercross or backcross progeny from a cross of divergent zebrafish strains is straightforward. The generation of mapping panels from haploid embryos is more complex; however, these data sets do not have heterozygous progeny, and the assignment of genotypes can, in many instances, be more conclusively scored (see Fig. 1). Before a primer pair is selected for SSCP mapping analysis, it is tested for a polymorphism in parental DNA of the strains used to generate the mapping panel. It is often necessary to test several primer pairs (which ideally are nonoverlapping) to find one with an easily resolved polymorphism. If a scorable polymorphism is found, the mapping panel is tested, and the allele distribution for the gene locus is determined. The Mapmanager software (Manly, 1993; available at http//:mcbio.med.buffalo.edu/mapmgr.html) is highly recommended for recording genotype distribution data and for determining linkage. Originally developed for genetic mapping in the mouse, it is useful for the analysis of genetic mapping experiments using backcrosses, intercrosses, recombinant inbred (RI) strains, or radiation hybrids. It facilitates the storage, retrieval, and display of genotype data and includes tools for statistical analysis of the experimental results. Of particular utility is a simple interface for positioning new genes on a reference cross. There are a number of potential difficulties in this procedure about which the investigator should be aware. In theory, a polymorphic fragment can be found in any expressed sequence as long as sufficient noncoding sequence is available. The efficiency of finding a polymorphic allele depends, to a considerable degree, on the size of the screened DNA region. If the 3’UTR is large enough, multiple primer pairs may be designed (which should ideally test minimally overlapping
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sequences) and tested for polymorphisms,or the use of intron or 5’UTR sequence can be employed for primer design. Discerning whether an apparent SSCP is “real” can require some experience. It is not uncommon that minor bands will be variably detectable, and their presence may be incorrectly assessed as being strain-specific. A simple means to reduce this source of error is to perform duplicate reactions using the parental DNAs of the mapping cross and to load these samples in alternating sequence. A true strain-specific shift can generally be readily recognized. Complexity of the SSCP patterns may cause problems. In some cases, multiple independently segregating products are found for one primer pair. Again, testing several PCR primer combinations derived from a sequence of interest may clarify which polymorphism corresponds to the locus. Alternatively, it is possible to elute a band identified as polymorphic, reamplify the fragment, and determine its sequence. In summary, SSCP mapping is an efficient tool to localize transcribed sequences in the zebrafish genome. Moreover, SSCP mapping is not restricted to fully characterized genes but can be used for localization of ESTs, similar to the efforts in human (Adams et al., 1991) and mouse (Brady et al., 1997). Because SSCP analysis can be used to map any unique genomic sequence, it is furthermore useful for analyzing sequences derived from the ends of large-insert clones, such as BACs, PACs, or YACs. For example, to assess possible chimerism in these clones, one can test that both ends colocalize. Additionally, in a chromosomal walk to a establish a contig over a region of interest, SSCP analyses may be helpful to determine the orientation of large-insert clones.
IV. Methods A. Primer Labeling 1. Sufficientsignal strength can obtained by labeling only one of the oligonucleotide primers, although maximal polymorphism detection is facilitated by labeling both. One microliter per reaction of a labeled primer mix prepared as follows will be used. The example is sufficient for 30 reactions: 24.2 p1 3.0 p1 1.0 p1 1.0 pl 1.2 p1 0.6 pl
dH2O 1OX T4 polynucleotide kinase buffer2 20pM forward primer 20pM reverse primer Y2P dATP, 6000Ci/mmole, 10mCilpl T4 polynucleotide kinase (10 ulpl)
2. Incubate at 37°C for 35-45 minutes. 1OX polynucleotide kinase buffer: 0.5M Tris-HCI pH 7.6; 0.1M MgC12; 0.05M Dithiothreitol; 1mM Spermidine-HCI; 1mM EDTA pH 8.0.
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3. Heat inactivate the reaction at 68°C for lominutes. 4. Keep reaction on ice until needed or store at -20°C. B. PCR Reaction
1. All PCR reactions are carried out in 96-well plates with a final reaction volume of 12.5 p1, and 1pl of template DNA (16-50 ng) is added to each well. For mapping panels that are to be analyzed for many markers, it is convenient to aliquot template DNA into plates and allow it to air-dry. These plates can then be stored. The dried DNA is readily rehydrated during PCR. 2. Use 9.25 p1 per reaction of a PCR master mix prepared as follows. The example is sufficient for 30 reactions: 205 p1 37.5 pl 2.5 pl 2.5 pl 30 pl
dH2O 1OX PCR buffer3 20pM forward primer 20p.M reverse primer ~ ~ ~ P - l a b eprimer l e d mix
3. Add 9.25 pl of the PCR master mix to each DNA template. 4. Mix well and overlay with 30 pl light mineral oil. 5. Mix on ice 0.5 p1 of 10mM each dNTP (dATP, dCTP, dGTP, dTTP) and 1 unit of Taq polymerase for each PCR. 6. Perform hot-start PCR: Denature the PCR reaction at 94°C for 5 minutes; hold at 80°C. 7. Add 2.25 p1 of dNTP/Taq mix to each reaction; mix well. 8. Amplify using appropriate thermocycling conditions for the primers used in the reaction. Our standard PCR conditions follows:
29-40 cycles: annealing: extension: denaturing: final cycle: annealing: extension:
55°C for 2 minutes 72°C for 3 minutes 94°C for 1 minutes 55°C for 2 minutes 72°C for 7 minutes
9. Store PCR at -20°C or keep on ice until needed. C. SSCP Gel Electrophoresis
1. Prepare gel mix for 5% polyacrylamide SSCP gel. Prepare 80 ml (sufficient for the Gibco BRL S2 sequencing gel apparatus used with a 0.4-mm spacer) as follows: 1OX PCR buffer: 0.1M Tris-HC1 pH
8.0;0.5M KCI; 0.02M MgC12; 0.01% gelatin.
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66 ml 4 ml 10 ml 800 PI 30pl
dHzO 1OX TBE (final concentration = OSX) acrylamide mix (38% acrylamide:2% Bis-acrylamide) 10% APS (ammoniumperoxodisulfate) in HzO, freshly prepared TEMED
2. Pour SSCP gel, insert comb, clamp well, and allow to polymerize for 2 hours or overnight. Avoid any bubbles in the gel. 3. Pre-equilibrate polymerized gel in gel apparatus with 0.5X TBE for at least 30 minutes at 4°C. 4. For each reaction, mix 2 p1 PCR product and 4 p1 USB stop solution solution (95% formamide; 0.05% bromphenol blue; 0.05% xylene cyanol; 0.02M EDTA pH 8.0) in a 96-well plate. Denature at 94°C for 5 minutes, transfer immediately onto ice. 5. Load 3-5 p1 per reaction onto gel. Run the gel for 90-150 minutes (according to the size for PCR product) in 0.5X TBE at 4°C and 40 W with constant power. 6. Transfer the gel onto Whatman filter paper, cover with plastic wrap, and dry on a gel dryer for 30 minutes. 7. Expose the gel to X-ray film overnight; do not use amplifying screens. References Adams, M., Kelley, J., Gocayne, J., Dubnick, M., Polymeropoulos, M., Xiao, H., Merril, C., Wu, A., Olde, B. and Moreno, R. (1991). Complementary DNA sequencing: Expressed sequence tags and human genome project. Science 252, 1651-1656. Beier, D. R., Dushkin, H. and Sussman, D. J. (1992). Mapping genes in the mouse using singlestrand conformation polymorphismanalysis of recombinant inbred strains and interspecificcrosses. Proc. Natl. Acad. Sci. U.S.A. 89, 9102-9106. Beier, D. R. (1993). Single-strand conformation polymorphism (SSCP) analysis as a tool for genetic mapping. Mamm. Genome 4,627-631. Brady, K. P., Rowe, L. B., Her, H., Stevens, T. J., Eppig, J., Sussman, D. J., Sikela, J., and Beier, D. R. 1997. Genetic mapping of 262 loci derived from expressed sequences in a murine interspecific cross using single-strand conformational polymorphism analysis. Genome Res. 7, 1085-1093. Driever, W., Solnica-Krezel,L., Schier, A. F., Neuhauss, S. C. F., Malicki, J., Stemple, D. L., Stainier, D. Y. R., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J. and Boggs, C. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123,37-46. Foernzler, D., Her, H., Knapik, E. W., Clark, M., Lehrach, H., Posthlethwait, J. H., Zon, L. I., and Beier, D. R. (1998). Gene mapping in zebrafish using single-strand conformation polymorphism analysis. Genomics, in press. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eden, F. J. M., Jiang, Y.-J., Heisenberg, C.-P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C. and Niisslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36. Johnson, S. L., Gates, M. A., Johnson, M., Talbot, W. S., Home, S., Baik, K., Rude, S., Wong, J. R., and Posthlethwait,J. H. (1996). Centromere-linkage analysis and consolidation of the zebrafish genetic map. Genetics 142,1277-1288.
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Knapik, E. W., Goodman, A., Atkinson, 0. S., Roberts, C. T., Shiozawa, M., Sim, C. U., WekslerZangen, S., Trolliet, M. R., Futrell, C., Innes, B. A,, Koike, G., McLaughlin, M. G., Pierre, L., Simon, J. S., Vilallonga, E., Roy, M., Chiang, P.-W., Fishman, M. C., Driever, W., and Jacob, H. J. (1996). A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development 123,451-460. Lincoln, S. E., Daly, M. J., and Lander, E. S. (1991). PRIMER: A computer program for automatically selecting PCR primers. Available from [email protected]. Manly, K. F. (1993). A Macintosh program for storage and analysis of experimental genetic mapping data. Mamm. Genome 4,303-313. Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5,74-879. Soto, D., and Sukumar, S. (1992). Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA. PCR Methods Applic. 2, 96-98.