Use of SNPs to determine the breakpoints of complex deficiencies, facilitating gene mapping in Caenorhabditis elegans
© Kadandale et al; licensee BioMed Central Ltd. 2005
Received: 17 February 2005
Accepted: 26 May 2005
Published: 26 May 2005
Genetic deletions or deficiencies have been used for gene mapping and discovery in various organisms, ranging from the nematode Caenorhabditis elegans all the way to humans. One problem with large deletions is the determination of the location of their breakpoints. This is exacerbated in the case of complex deficiencies that delete a region of the genome, while retaining some of the intervening sequence. Previous methods, using genetic complementation or cytology were hampered by low marker density and were consequently not very precise at positioning the breakpoints of complex deficiencies. The identification of increasing numbers of Single Nucleotide Polymorphisms (SNPs) has resulted in the use of these as genetic markers, and consequently in their utilization for defining the breakpoints of deletions using molecular biology methods.
Here, we show that SNPs can be used to help position the breakpoints of a complex deficiency in C. elegans. The technique uses a combination of genetic crosses and molecular biology to generate robust and highly reproducible results with strong internal controls when trying to determine the breakpoints of deficiencies. The combined use of this technique and standard genetic mapping allowed us to rapidly narrow down the region of interest in our attempts to clone a gene.
Unlike previous methods used to locate deficiency breakpoints, our technique has the advantage of not being limited by the amount of starting material. It also incorporates internal controls to eliminate false positives and negatives. The technique can also easily be adapted for use in other organisms in which both genetic deficiencies and SNPs are available, thereby aiding gene discovery in these other models.
Genetic complementation results for sDf62, showing it to be complex
3.99 ± 0.003
4.58 ± 0.001
4.66 ± 0.002
4.67 ± 0.033
4.81 ± 0.011
4.81 ± 0.024
4.81 ± 0.035
4.82 ± 0.017
4.82 ± 0.017
4.82 ± 0.033
4.82 ± 0.051
4.82 ± 0.069
4.83 ± 0.042
4.86 ± 0.069
4.96 ± 0.069
4.97 ± 0.069
5.00 ± 0.046
5.18 ± 0.003
5.43 ± 0.012
5.51 ± 0.038
5.73 ± 0.035
5.75 ± 0.053
5.78 ± 0.037
6.01 ± 0.016
6.13 ± 0.083
7.97 ± 0.010
Another method that could be used to achieve the same result is Southern Blotting . However, this method is tedious, involving the preparation of high molecular weight genomic DNA, running gels and probe hybridization, and requires a larger starting amount of DNA.
In human research, taking advantage of the growing number of SNPs identified in the human genome, approaches are being developed to use SNPs to identify regions of loss of heterozygosity (LOH), and/or deletions in chromosomes . The availability of a strain of C. elegans, CB4856, which is predicted to harbor about 1 SNP per kilobase of genomic sequence [9, 10] when compared to the wild-type strain (N2), led us to believe that we could adopt similar methods for use in C. elegans, thereby overcoming the limitations of using deficiencies in this model system. In fact, the C. elegans SNP database already contains about 6,000 confirmed and predicted SNPs between the N2 and CB4856 strains , which corresponds to a SNP density of about 1 per 15 Kb of sequence. As the use of SNPs to map genes increases [12, 13], we expect that many more SNPs will be discovered, making their use even more attractive. We report here the use of SNP mapping, in conjunction with traditional genetic deletion mapping to obtain a better map of the breakpoints of a complex deficiency, thereby greatly assisting the process of gene mapping and characterization.
Results and Discussion
Identification of ozDf2 as a complex deficiency
Use of SNPs to map the breakpoints of ozDf2
We have shown here that using SNPs as genetic markers in model systems greatly eases the problems of defining the breakpoints of deficiencies, both simple and complex. This allows us to use them as powerful tools in narrowing down the region of interest when cloning genes.
Our use of a combination of genetic complementation data along with SNP mapping of deficiency breakpoints has allowed us to rapidly narrow down the region of interest for cloning spe-19. Deficiency mapping in a number of organisms, including C. elegans, has been beset by the lack of a convenient method to correlate genetic complementation data with the physical map, confounding our ability to accurately define the breakpoints of deficiencies. Previous methods used to overcome this obstacle depended on a negative result [6, 7], which is often untrustworthy. Using SNPs, not only is the correlation between the genetic and physical maps easily achieved, but we can also, potentially, generate higher resolution maps of the breakpoints of both simple, as well as complex deficiencies. Additionally, the amplification of the identical locus from the CB4856 chromosome serves as a good internal control, eliminating false positives and false negatives and allowing us to interpret our data with a very high degree of confidence. The fact that we can use heterozygous animals, without the need to generate homozygous, dead embryos overcomes the limitations of previous methods which relied exclusively on negative results, such as using PCR to detect the presence or absence of a sequence. We chose to use bulk worm lysates from a heterozygous line as our source of DNA. However, the technique would work just as well if single worms (after confirming their genotype) were used as the source of DNA. We believe that using lines has the following advantages:
1. The line can be frozen away and then rethawed at a later date if further analysis is required.
2. Multiple SNPs can be tested using the same lysate, as opposed to requiring multiple, single worm lysates.
3. Lysates can be prepared in bulk, in replicates, for independent and robust confirmation of all results obtained by this method.
4. No special care needs to be taken for preparation of DNA for SNP detection. DNA is made from the lines using a simple, scaled up version of the method used to make worm lysates for single worm PCR.
We initially chose to use SNPs that caused an abrogation of a restriction enzyme recognition site in the CB4856 sequence, thereby eliminating any artefacts due to incomplete restriction enzyme digestion. However, by running a control reaction to ensure that enzyme digestion has progressed to completion, one could also use SNPs that cause the loss of a restriction enzyme recognition site in the N2 sequence. Additionally, this method allows the use of SNPs that are only different in sequence, causing no changes in restriction enzyme recognition sites. A simple sequencing reaction can be done to check whether the Deficiency/CB4856 worms had only CB4856 sequence (which would be the case if the deficiency deletes the region of the SNP), or whether both N2 and CB4856 sequence was present (indicating that the SNP region is not lost in the deletion being tested). In addition to the CB4856 strain which we have used, other wild isolates of C. elegans which harbour SNPs  can also be used, expanding the possibilities of this technique.
While we have shown this method to work in the model system C. elegans, a similar approach can be applied in any organism where deficiencies/deletions exist and where SNP mapping is possible.
Culturing of C. elegans strains
Worms were grown and cultured according to standard protocols described elsewhere . The strains used for this work were: spe-19, ozDf2/dpy-21 par-4, N2 (the wild-type) and CB4856.
Survey of deficiencies on WormBase
We looked at the complementation data for 20 deficiencies chosen at random. We ensured that deficiencies from all chromosomes were included in the analysis and that the deficiencies chosen had their left and right boundaries defined by cloned genes (this allowed us to set the other boundaries of the deficiency with certainty). We then looked at the complementation data and scored the deficiency as "complex" if at least one of the genes that were within the outer boundaries of the deficiency was complemented by the deficiency. While scoring the complex deficiencies, we made sure that the internal locus that was complemented was either cloned or had multiple, corroborating mapping data. If the deficiency failed to complement all the loci that were within the outer boundaries, it was scored as a simple deficiency. The deficiencies that we scored as simple are: nDf20, sDf10, mnDf16, nDf3, tDf1, mnDf1, ccDf1,eDf2, tDf5, ctDf2 and ctDf3. The complex deficiencies we found are: hDf10, hDf17, nDf19, nDf9, eDf18, qDf8, eDf3, maDf4 and nDf27.
Genetic complementation test of ozDf2
Since spe-19 males are fertile, we mated ozDf2/dpy-21 par-4 with spe-19 males. Of the outcross F1 progeny, we would expect half to be of the genotype ozDf2/spe-19 and consequently, sterile. The other half would be fertile, spe-19/dpy-21 par-4 worms. However, all the F1 progeny were fertile, indicating that the deletion ozDf2 complemented spe-19, belying our expectations. The presence of the deletion and of spe-19 was confirmed by looking at the F2 progeny. Half the F1 progeny segregated wild-type animals, dead eggs and sterile worms, indicating that their genotype must be ozDf2/spe-19. Thus, ozDf2 complements spe-19.
Generation of lines for SNP mapping of the breakpoints of ozDf2
We crossed ozDf2/dpy-21 par-4 hermaphrodites to CB4856 males. These ozDf2/dpy-21 par-4 heterozygotes are wild-type, but ozDf2 homozygous animals are embryonic lethal and dpy-21 par-4 homozygotes are short and fat – a phenotype referred to as Dumpy, or Dpy. F1 animals that segregated Dpy progeny were discarded (these would have been dpy-21 par-4/CB4856), and only the remaining, heterozygous animals (which are all ozDf2/CB4856) were used to set up lines that were further analyzed for their SNP sequences (Figure 5). Individual ozDf2/CB4856 worms were picked to separate culture plates. These worms are fertile and generate independent lines which were used for further analysis. Each line was generated within 6 days of picking the founder worm, ensuring that the N2 deficiency chromosome would not be underrepresented significantly in the line. Thus, each individual line would contain a mixture of CB4856 and ozDf2 chromosomes, which provide abundant material for the analysis of multiple SNP sequences. We established three independent heterozygous lines for analysis.
SNP mapping of the breakpoints of ozDf2
Crude genomic DNA was prepared from the three ozDf2/CB4856 lines by methods described previously . We used the Vector NTI suite to design primers that amplified ~500 bp upstream and downstream of the SNP, resulting in a PCR product that was approximately 1 Kb. The details of the primers used and the PCR products generated are as follows:
SNP Y113G7A:29 – The expected product size is 1033 bp using the forward primer (GTAGGAAGTGGTGCCAGATGACGAGG) and the reverse primer (GTCTCAATTTGTGAGCGCATGTC).
SNP F38A6:14347 – The expected product size is 961 bp using the forward primer (TGGTTCCCCTCTACCAGATGCC) and the reverse primer (CCAACACAACTCAATCTAATGTTTACC).
We used these primers to amplify the corresponding product from each of the three genomic DNA preparations of the ozDf2/CB4856 lines using the following PCR conditions: denature at 94°C for 30 seconds, anneal at 60°C for one minute, extend at 72°C for 1 minute and repeat for 30 cycles. The PCR product was then digested with an appropriate restriction enzyme, and the products analysed on a 1.5% agarose gel run using the SB buffer. For SNP Y113G7A:29, Xmn I was used to digest the PCR product and for SNP F38A6:14347, Dra I was used.
This work was supported by grants from the NIH (R01 GM63089-01) and the Johnson & Johnson Discovery Award. PK was supported by the Charles and Johanna Busch pre-doctoral fellowship. The Caenorhabditis Genetics Center provided some nematode strains and it is funded by the NIH National Center for Research Resources (NCRR).
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