An accurate method for quantifying and analyzing copy number variation in porcine KIT by an oligonucleotide ligation assay.
BACKGROUND
Aside from single nucleotide polymorphisms, copy number variations (CNVs) are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing, real-time PCR, invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed.


RESULTS
PCR followed by a quantitative oligonucleotide ligation assay (qOLA) was developed for quantifying CNVs. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares of bias and standard deviation of qOLA were 2.09 and 0.45, respectively. These values are less than half of those in the published pyrosequencing assay for analyzing CNV in porcine KIT. Using a combined method of qOLA and another pyrosequencing for quantitative analysis of KIT copies with spliced forms, we confirmed the segregation of KIT alleles in 145 F1 animals with pedigree information and verified the correct assignment of genotypes. In a diagnostic test on 100 randomly sampled commercial pigs, there was perfect agreement between the genotypes obtained by grouping observations on a scatter plot and by clustering using the nearest centroid sorting method implemented in PROC FASTCLUS of the SAS package. In a test on 159 Large White pigs, there were only two discrepancies between genotypes assigned by the two clustering methods (98.7% agreement), confirming that the quantitative ligation assay established here makes genotyping possible through the accurate measurement of high KIT copy numbers (>4 per diploid genome). Moreover, the assay is sensitive enough for use on DNA from hair follicles, indicating that DNA from various sources could be used.


CONCLUSIONS
We have established a high resolution quantification method using an oligonucleotide ligation assay to measure CNVs, and verified the reliability of genotype assignment for random animal samples using the nearest centroid sorting method. This new method will make it more practical to determine KIT CNV and to genotype the complicated Dominant White/KIT locus in pigs. This procedure could have wide applications for studying gene or segment CNVs in other species.
Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea. seoboyeong@hanmail.net
Bo-Young
BY
Seo
Eung-Woo
EW
Park
Sung-Jin
SJ
Ahn
Sang-Ho
SH
Lee
Jae-Hwan
JH
Kim
Hyun-Tae
HT
Im
Jun-Heon
JH
Lee
In-Cheol
IC
Cho
Il-Keun
IK
Kong
Jin-Tae
JT
Jeon
D000818:Animals; D005260:Female; D018628:Gene Dosage; D020440:Gene Duplication; D005838:Genotype; D006200:Hair Color / Q000235:genetics; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D034421:Sus scrofa / Q000235:genetics*