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.
The samples of DNA were obtained by plucking individual hairs from students' heads and using the PCR device to replicate. the DNA from the roots of the hair. The replicated DNA samples were then placed into the electrophoresis gel and the device was turned on. Using the methods discussed above we found that three of the fourteen samples, 21%, were homozygous and the remaining eleven samples. 79% were heterozygous in their ancestors.
In our genes, multiple different alleles determine whether one person will have a certain trait or not. Alleles are what make-up our genotypes and in this lab, we wanted to determine the genotypes of our class in the two loci: TAS2R38 and PV92. The TAS2R38 locus codes for a protein that involves the bitter taste of PTC; the gene determines whether or not a person will taste the PTC paper as very bitter or no taste at all. People with the “T” allele are tasters while those that are homozygous recessive (tt) are non-tasters. The taster locus can be found chromosome 7.3 The two different alleles present in the could be due to the effect of evolution and natural selection because the same can be found in chimps.4 The PV92 locus does not code for any protein but rather involves an Alu element that is 300-bp long. A person with the “+” allele would have the Alu element making that sequence longer while those with the “-“ allele don’t have the element and would have a shorter sequence. This locus can be found on chromosome 16.3 There are multiple Alu sequences found among primate genomes but there are human specific sequences such as the one found on the PV92 locus.1 In the experiment, student DNA was collected from cheek cells and PCR was used to target the loci and amplify the region of DNA. In the taster gene, after amplification, a restriction digest was performed to differentiate between the two alleles. The digest was able to show differentiation because those with the “T” allele would have two bands from gel electrophoresis and those with “t” will have one band because the restriction enzyme doesn’t cut it. For the PV92, we were able to distinguish between the alleles due to the added length of the Alu element. Those...
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