[논문]Quantitative evaluation of the molecular marker using droplet digital PCR

Shin, Wonseok; Kim, Haneul; Oh, Dong-Yep; Kim, Dong Hee; Han, Kyudong

doi:10.5808/gi.2020.18.1.e4

[국내논문] Quantitative evaluation of the molecular marker using droplet digital PCR 원문보기

Genomics & informatics, v.18 no.1, 2020년, pp.4.1 - 4.6

Shin, Wonseok (Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University) , Kim, Haneul (Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University) , Oh, Dong-Yep (Livestock Research Institute) , Kim, Dong Hee (Department of Anesthesiology and Pain Management, Dankook University College of Medicine) , Han, Kyudong (Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University)

Abstract ▼ AI-Helper

Transposable elements (TEs) constitute approximately half of Bovine genome. They can be a powerful species-specific marker without regression mutations by the structure variation (SV) at the time of genomic evolution. In a previous study, we identified the Hanwoo-specific SV that was generated by a TE-association deletion event using traditional PCR method and Sanger sequencing validation. It could be used as a molecular marker to distinguish different cattle breeds (i.e., Hanwoo vs. Holstein). However, PCR is defective with various final copy quantifications from every sample. Thus, we applied to the droplet digital PCR (ddPCR) platform for accurate quantitative detection of the Hanwoo-specific SV. Although samples have low allele frequency variation within Hanwoo population, ddPCR could perform high sensitive detection with absolute quantification. We aimed to use ddPCR for more accurate quantification than PCR. We suggest that the ddPCR platform is applicable for the quantitative evaluation of molecular markers.

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가설 설정

In addition, the qPCR has the following disadvantages. (1) The accuracy of qPCR depends on proper experimental design based on well-established reference genes. (2) For absolute quantification, you should create a standard curve for data normalization based on changes in the transcription level of the reference gene [16].
The absolute copy number of Hanwoo samples were approximately two times less than that of Holstein samples. (B) The VIC fluorescence was only detected strongly in Hanwoo samples.

제안 방법

This study uses ddPCR, the Stilla Naica System for Crystal Digital PCR, to overcome the limitations of PCR and to accurately evaluate the Hanwoo-specific SV locus that was identified in the previous study [7]. We suggest that the ddPCR platform can be used as a quantitatively and numerically sensitive method with molecular markers.
Prior to the experiment, we confirmed the quantification of Hanwoo and Holstein DNAs using Qubit 4.0 Fluorometer (Thermo Fisher Scientific) with 1 × dsDNA HS (high-sensitivity) assay kit (Thermo Fisher Scientific) for dsDNA measurement.
1B). DNA templates from five Hanwoo and five Holstein blood samples were conducted to the ddPCR assay with designed primer/ probe sets. The extracted DNAs should be assessed for accurate quantification using a UV spectrophotometer (NanoDrop, Thermo Fisher Scientific) and an intercalating reagent reaction with the dsDNA (Qubit assay).

참고문헌 (26)

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[국내논문] Quantitative evaluation of the molecular marker using droplet digital PCR 원문보기

Abstract ▼ AI-Helper

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[국내논문] Quantitative evaluation of the molecular marker using droplet digital PCR 원문보기

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