Paste yChondraah Holmes
Dr. Julie Hall
Cell Biology & Genetics
18 March 2016
Rapid single nucleotide polymorphism mapping in C. elegans Technique Review
SNP Mapping Related to the Literature Article
Single Nucleotide Polymorphisms (SNPs) are any type of variation that can occur in the genome (Jabukowski & Kornfeld, 1999). SNP mapping is a well-recognized technique within the field of C. elegans research (Zipperlen et al., 2005, Swan, Curtis, McKusick, Volnov, Mapa, & Cancilla, 2002, Kaletta & Hengartner, 2006). The paper began by briefly explaining the two advantages of utilizing SNP mapping (Davis, Hammarlund, Harrach, Hullett, Olsen, & Jorgensen, 2005). The first being that there is no associated phenotype, meaning that mutations that are masked by other typical marker mutations can still be mapped without being affected. Secondly, SNPs are more closely compacted compared to other markers. Because of this, SNP mapping could potentially offer the resolution of single genes. These two advantages make SNP mapping an attractive technique for C. elegans researchers (Davis et al., 2005). The purpose of using this method in this article was to show that SNP mapping could be used to correctly map a known gene, dpy-5. Furthermore, they used this method to map the mutations in an undifferentiated strain, and to show that the behavioral phenotype for that particular strain can be mapped to three loci at the same time (Davis et al., 2005). SNP mapping is typically done in two phases (Davis et al., 2005). The first is chromosome mapping, which is similar to the customary two-factor mapping, which occurs when a mutation in the gene of interest is mapped against a specific marker mutation. This is used to assign various mutations to different individual. The primary goal of chromosome mapping is to classify the pertinent chromosome and to gain a rough idea of the position of that particular gene of interest (Davis et al., 2005). The second phase of this method is interval mapping, which tries to place the gene of interest that was previously found within a small break in between two SNPs in order to fine tune the location of the gene. The traditional SNP detection method incorporates using snip-SNPs, which are SNPs that can be identified using Restriction Fragment Length Polymorphism (RFLP) as a direct result of utilizing restriction enzyme recognition sites (Davis et al., 2005). Even though other methods have been used for mapping of C. elegans including fluorescence polarimetry and indel detection, which look for insertion and deletion mutations, snip-SNPs are very easy to consider because they do not require as much investment or advanced equipment (Swan et al., 2002, Zipperlen et al., 2005). However, the one disadvantage of using this procedure was requirement of PCR amplification, which could be a very tedious task (Davis et al., 2005). Davis et al. utilized a faster and more efficient form of SNP mapping that would be more effective, more plentiful, and also more accurate which makes it very attractive for many C. elegans researchers (2005).
Origins of the Technique
The technique and concept of genetic mapping have been around for over a century and has allowed for the creation of various types of mapping. Since its inception in 2001, SNP mapping, a modification of a standard or older technique, has become the most commonly used form of mapping for genetic linkage in C. elegans (Davis et al., 2005, Doitsidou, Poole, Sarin, Bigelow, & Hobert, 2010). However, Jabukowski & Kornfeld first used SNP mapping as a method to clone genes in C. elegans in 1999 (Strange, 2006). Additionally, SNP mapping against a particular polymorphic strain from Hawaii, CB4856, was discovered in recent years (Wicks et al., 2001). When using this procedure, the wild-type C. elegans strain (N2 Bristol) and the previously mentioned CB4856 are used as genetic markers (Davis et al., 2005).
Mapping with SNPs has become a strong complement and in some cases a complete alternative to the standard genetic mapping protocols. When considering the use of SNP mapping, three other techniques should be investigated. Firstly, transposons, which are DNA fragments that hop around the genome, can be used as mutagens (Hoskins et al., 2001). Inserting a transposon in a place where it does not typically belong can mutate a gene and tag it at the same time (Strange, 2006, Hoskins et al., 2001). The genes that are identified in forward transposons can be cloned without the use of mapping. Secondly, developing RNAi feeding libraries allows whole-genome screens to be utilized. This method eliminates the use of mapping because the target of each molecular RNAi is known. Additionally, when the mutations are created by the reverse genetic procedures, mapping is not a necessity (Strange, 2006). Even though all of these techniques may provide valuable data, chemical mutagenesis, the chemically induced genetic changes in DNA structure that can affect multiple genes, is the easiest way to create mutants. Moreover, SNP mapping allows this process to be accomplished effectively (Strange, 2006). An added benefit of SNP mapping is that it has not just been used in C. elegans. Other model organisms including, Drosophila melanogaster have been used for this technique (Hoskins et al., 2001). Various scientists have made numerous changes to SNP mapping methods. These improvements have made SNP mapping a strong and efficient procedure for identifying and cloning a particular gene of interest in various organisms.
Technique Methodology
The model organism of choice for this study was C. elegans. In order to begin mapping a mutation onto a chromosome, the authors conducted some modifications to the standard technique in order to increase the speed and to decrease the potential error that may occur in the procedure. They hoped to do this by simplifying the procedure and performing all of the steps in a 96-well plate, and designing the experiment so that every SNP reaction could be completed under the same conditions (Davis et al., 2005). In order to accomplish this, they had to create primers that used the same conditions for polymorphism detection and the primers had to all use the same conditions for magnification. It is important to note that SNPs are typically located in the non-coding regions of the DNA that are rich in A-T nucleotides. Moreover, because of these A-T rich regions, the PCR primer has to anneal to that region to get amplification at the spot of interest, which provoked them to use the enzyme Dra1 for good coverage. As aforementioned, SNP mapping occurs in two phases with the first phase being chromosome mapping. During this portion, 30 mutant animals (homozygous Bristol DNA around the mutation) and 30 wild-type animals (heterozygous Bristol/Hawaiian or homozygous Hawaiian DNA) were broken apart in 20 μL of lysis buffer, which was done so that the cells could be lysed and their compounds could be analyzed. The lysate was then added to a PCR mix that did not have any primers, and this mix were put into every other row on the 96-well plate. The primers were added to the master plate using pin replication. Each mutant was placed next to the control based on the loading mechanism of the 8-channel pipette. After amplification occurred, the PCR products were digested utilizing the Dra1 enzyme, and were eventually loaded onto an agarose gel. This gel showed all 48 of the SNP markers.
The next portion of the procedure included interval mapping in order to fine-tune the location of the gene after they determined the rough position of the mutation on the chromosome. This part of the procedure was different from chromosome mapping in that individual mutant genotypes are able to be determined in contrast to the genotype of the collective group of animals. Recombinants of the animals were taken from the heterozygotes or any delegate of the offspring. These were placed in the wells of a 96-well PCR plate and then broken down. After this occurred the broken down DNA was pin replicated into a master PCR mix that contained primers for SNP that was of interest at the moment. These plates were then processed for PCR amplification, digested with the DraI enzyme, and then ran on an agarose gel. No explicit data analysis or statistical methods were mentioned.
Future Directions of Applications
SNP mapping has allowed great advances in the field of science including the ability to map mutations in a quick way. However, there is always room for improvement and other directions that can be taken in the future. One of the biggest challenges facing evolutionary biologists is being able to understand gene loci that may explain fitness variation in various populations (Slate et al., 2009). Perhaps, adapting new or more advanced portions of the technique will allow for increased understanding of how genes interact with the environment. Another future direction could make SNP mapping more efficient in regards to coding regions within the DNA. SNPs that are found in non-coding regions can lead to higher risk of cancer, and therefore may affect the structure of mRNA and how likely an individual is to develop the disease. SNP mapping provides an interest of gene therapy and specializing the technique to accommodate those non-coding regions of the DNA in order to combat this issue. Regardless of what future methods and research designs that continue to be developed, it is great to know that innovative and monumental efforts are being made and discovered in C. elegans research.
References
Chakravarti, A. (2001). Single nucleotide polymorphisms:…to a future of genetic medicine. Nature, 409, 822-823.
Davis, M.W., Hammarlund, M., Harrach, T., Hullett, P., Olsen, S., & Jorgensen, E.M. (2005). Rapid single nucleotide polymorphism mapping in C. elegans. BMC Genomics, 6,118.
Doitsidou, M., Poole, R. J., Sarin, S., Bigelow, H., & Hobert, O. (2010). C. elegans mutant Identification with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS ONE, 5(11), 1-7.
Hoskins, R. A., Phan, A.C., Naeemuddin, M., Mapa, F.A., Ruddy, D.A., Ryan,. J.J., Young, L.M., Wells, T., Kopczynski, C., & Ellis, M.C. (2001). Single Nucleotide Polymorphism Markers for Genetic Mapping in Drosophila melanogaster. Genome Research, 11(6), 1100-1113.
Jakubowski, J., & Kornfeld, K. (1999). A local, high-density, single-nucleotide polymorphism map used to clone Caenorhabditis elegant cdc-1. Genetics, 153, 743-752.
Kaletta, T., & Hengartner, M. O. (2006). Finding function in novel targets: C. elegans as a model organism. Nature Reviews Drug Discovery, 5(5), 387-399.
Sarin, S., Prabhu, S., O'meara, M. M., Pe'er, I., & Hobert, O. (2008). Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nature Methods, 5(10), 865 867.
Slate, J., Gratten, J., Beraldi, D., Stapley, J., Hale, M., & Pemberton, J.M. (2009). Genetic mapping in the wild with SNPs: guidelines and future directions. Genetica,136, 97-107.
Strange, K. (2006). C. elegans: Methods and applications. Totowa, NJ: Humana Press.
Swan, K. A., Curtis, D. E., McKusick, K. B., Volnov, A. V., Mapa, F. A., & Cancilla, M. R. (2002). High-throughput gene mapping in Caenorhabditis elegans. Genome Research, 12, 1100-1105.
Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H., & Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nature Genetics, 28, 160-164.
Zipperlen, P., Nairz, K., Rimann, I., Basler, K., Hafen, E., Hengartner, M., & Hajnal, A. (2005). A universal method for automated gene mapping. Genome Biology, 6, R19.
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