Correcting for Errors Inherent in DNA Pooling Methods :: Exploratory Essays Research Papers

Correcting for Errors Inherent in DNA Pooling Methods Professor’s comment: This student’s research paper describes developments and refinements in an effort to correct for errors inherent in the DNA pooling methods employed in genetics research. I admire the clarity and efficiency with which she explains these developments. Introduction Many genetic diseases have yet to be located on the human genome for reasons that include their multiple loci and incomplete penetrance. To pinpoint these loci in terms of particular regions of the chromosomes, association studies, which compare allele frequency between affected individuals (probands) and controls, must be performed across the entire human genome. With approximately 0.4 cMs between markers, 10,000 microsatellite markers would be necessary to fully saturate the genome (Collins et al., 2000). For a study of 1000 probands and 1000 controls, 20 million genotypings would be required (Collins et al., 2000). Because of the relative impossibility of such a task, a technique called DNA pooling has been developed. DNA pooling involves the mixing of equal amounts of DNA from each individual in a group and then proceeding as one would with individual samples—by performing a PCR and running the samples out on a gel. DNA pooling can determine total allele content of a group, for one microsatellite, without the need to individually genotype each individual in that group. As such, it is an effective way to decrease the number of genotypings required, reducing the workload by factors of tens or hundreds. Although the benefits of DNA pooling are immense, two significant sources of error must be addressed. The first, known as stutter peaks, results from a slippage of the DNA polymerase during the PCR replication process. Stutter peaks appear as progressively smaller peaks before each real allele peak and result in artificially inflated values for the smaller allele sizes. This error is consistent and reproducible for a particular marker (Perlin et al., 1995). The other major source of error is caused by preferential amplification of some alleles over others. In this situation, uneven PCR replication and amplification result from the differing sizes of the fragments being replicated. Over the years, several methods have been developed to overcome these sources of error, making DNA pooling a practical method of screening for disease loci. Early Methods for Stutter Correction LeDuc et al. (1995) developed one of the first methods to correct for the stutter artifact by measuring the allele and stutter peak heights for the smallest allele of the pool and any allele not immediately adjacent to other alleles. Correcting for Errors Inherent in DNA Pooling Methods :: Exploratory Essays Research Papers Correcting for Errors Inherent in DNA Pooling Methods Professor’s comment: This student’s research paper describes developments and refinements in an effort to correct for errors inherent in the DNA pooling methods employed in genetics research. I admire the clarity and efficiency with which she explains these developments. Introduction Many genetic diseases have yet to be located on the human genome for reasons that include their multiple loci and incomplete penetrance. To pinpoint these loci in terms of particular regions of the chromosomes, association studies, which compare allele frequency between affected individuals (probands) and controls, must be performed across the entire human genome. With approximately 0.4 cMs between markers, 10,000 microsatellite markers would be necessary to fully saturate the genome (Collins et al., 2000). For a study of 1000 probands and 1000 controls, 20 million genotypings would be required (Collins et al., 2000). Because of the relative impossibility of such a task, a technique called DNA pooling has been developed. DNA pooling involves the mixing of equal amounts of DNA from each individual in a group and then proceeding as one would with individual samples—by performing a PCR and running the samples out on a gel. DNA pooling can determine total allele content of a group, for one microsatellite, without the need to individually genotype each individual in that group. As such, it is an effective way to decrease the number of genotypings required, reducing the workload by factors of tens or hundreds. Although the benefits of DNA pooling are immense, two significant sources of error must be addressed. The first, known as stutter peaks, results from a slippage of the DNA polymerase during the PCR replication process. Stutter peaks appear as progressively smaller peaks before each real allele peak and result in artificially inflated values for the smaller allele sizes. This error is consistent and reproducible for a particular marker (Perlin et al., 1995). The other major source of error is caused by preferential amplification of some alleles over others. In this situation, uneven PCR replication and amplification result from the differing sizes of the fragments being replicated. Over the years, several methods have been developed to overcome these sources of error, making DNA pooling a practical method of screening for disease loci. Early Methods for Stutter Correction LeDuc et al. (1995) developed one of the first methods to correct for the stutter artifact by measuring the allele and stutter peak heights for the smallest allele of the pool and any allele not immediately adjacent to other alleles.