Special Genotyping Applications

To identify susceptibility genes for complex disorders, large-scale association studies have been considered as a powerful approach. Because numerous SNPs must be tested in large numbers of individuals for this purpose, intensive efforts have been undertaken to develop efficient HT tools to further reduce genotyping costs. One suitable way to address the requirement of cost and time-effectiveness is automated and accurate genotyping as the application of DNA pooling (4).

Any pooling strategy relies on the accurate measuring of SNP allele frequencies in pools of cases and controls. Several technologies allowing accurate and reliable estimates of allele frequencies have been developed so far. Current available pooling methods are based on allele-specific primer extension, allele-specific PCR, DHPLC, BAMPER, and SSCP analysis.

Although the potential of DNA pooling for large-scale genotyping is obvious, several practical and theoretical issues have to be taken into account. To ensure reliability of any genotyping method, each step must be quantitative. The first critical step is the construction of DNA pools containing equal amounts of DNA from each individual sample. Therefore, some protocols use DNA-specific fluorimetric methods for quantification. Furthermore, the reliability of assays depends on the DNA quality of samples. Another common problem is that all currently used SNP detection assays initially require a target amplification. Many SNPs show unequal representation of both alleles due to different amplification efficiency. The bias in allelic representation that is often observed might also be caused by differential incorporation of nucleotides (in primer extension assays), differential efficiency of hybridization (in hybridization-based assays), and differential detection efficiency of allele-specific products [e.g., in mass spectrometry (MS) detection]. To yield real allele frequencies the bias in allele representation must be corrected by the factor estimated from heterozygotes. Because heterozygous individuals have an equal number of copies of both alleles, the ratio of signal strengths of one allele to the other allele reflects the bias in allelic representation. Ignoring unequal allele representation can result in biased tests of allelic association (5).

In order to benefit from pooling designs, especially in the analysis of complex diseases, it is an essential prerequisite that allele frequency estimates are highly reproducible. Several pooling designs have been proposed involving, either the number of replicate pools, the number of individuals pooled, or the number of distinct pools made of subsets of individuals. The effect of the pool size on the accuracy of frequency estimates appears to be negligible. Therefore, the use of larger pools and multiple replicates are recommended, reducing the amount of genotyping. Three to four replicates of each PCR and detection should be sufficient.

Comparing accuracy and reproducibility of SNP detection methods applied for pooling analysis primer extension-based strategies appear to achieve the most reliable results with standard deviations of 1% to 2% between replicates. Allele frequency estimates deviate from real frequencies by about 1% to 3%. In contrast, quantification of differences between case and control pools by allele-specific hybridization is hampered due to insufficient hybridization specificity. The common limiting factor of any available pooling method is that allele frequencies can consistently be detected in the range from 5% to 50%. Thus, pooling strategies are preferentially suitable for screening common variants that are thought to be the causative variants in complex diseases.

Considering the preferred application of pooling strategies to screen a genomic region with large numbers of SNPs, but rather small samples of pooled DNA, genotyping technologies with low assay set-up costs are favored. Even with highly accurate and reproducible methodologies, frequency estimation from DNA pools is prone to some kind of errors and loss of individual information. Therefore, the most effective use of DNA pooling might be a two-stage approach in which a dense set of markers should be tested first in pooled samples, followed by individual genotyping restricted to the most promising SNPs. In this way, DNA pooling designs can considerably reduce the amount of genotyping required, and hence, can be an effective strategy for systematic association studies.

Multiplexing is another approach to significantly increase HT without significantly increasing the costs. It means that multiple SNPs can be assayed simultaneously. The differences in multiplexing capacity of the current genotyping technologies are substantial. Although mass spectrometric detection of allele-specific primer extension products has been demonstrated, the potential of stable fivefold multiplex assays multiplexing with pyrosequencing appears to be more difficult in this magnitude. Hybridization-based assays with fluorescence detection multiplexing can be achieved by use of different fluorescent dyes. However, these methods need labor for assay optimization, which makes multiplexing only suitable for frequently repeated assays.

Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook

Post a comment