High-Resolution Melting analysis as a tool for rapid and sensitive detection of genotypes in cattle population

Transcription

1 Research and Development Station for Bovine, Arad, Romania High-Resolution Melting analysis as a tool for rapid and sensitive detection of genotypes in cattle population Daniela Elena Ilie, Ada Cean, Ioan Vintilă Simpozionul "Preocupări actuale și de perspectivă în genetica vegetală și animală" A.S.A. S. 20 iunie 2013

2 High Resolution Melting (HRM) Concept Target sequence amplified by PCR in the presence of a saturating fluorescent dye: ResoLight Dye After the PCR, amplicons are briefly denatured and then rapidly reannealed Finally, temperature is slowly increased (melting curve) The shape of the melting curve are different for homozygous and heterozygous samples This way, genetic variations (SNPs, mutations) are identified

3 HRM principle LightCyler480 ResoLight Dye bound to double-stranded DNA HRM characterizes double-stranded PCR products based on the melting behavior -Different DNA sequences melt at different specific temperatures This is measured in real time using intercalating dyes -The fluorescence decreases during DNA dissociation The HRM software plots fluorescence decrease vs. temperature

4 Fluorescent dye for HRM - Example: A to G variation Analysis software generating normalized and temperature shifted difference plot Normalized melting curves Difference plot - green=wildtype Variation in melting temperature - blue=mutant - red=heterozygote Amplicon Melting of homozygote samples (containing homoduplexes of wildtype or mutant DNA) give very similar curve shapes. Amplicon Melting of heterozygote samples (containing homo and heteroduplexes) give curve shapes which are highly distinct.

5 Analyzed genes Bovine leukocyte adhesion deficiency (BLAD) BLAD is a autosomal recessive disease that affects the Holstein cattle breed and is characterized by greatly reduced expression of the heterodimeric β2 integrin adhesion molecules on leukocytes, resulting in multiple defects in leukocyte function. BLAD is due to a single point mutation (A G), at position 128, resulting in a single amino acid change (aspartic acid glycine) in the β subunit (CD18) of the β2 integrin. BLAD is still a prevalent disease in cattle, with significant economic effect. Therefore, genetic screening is of utmost importance in order to obtain disease-free animals and to eliminate the mutant allele from the population. Kappa-casein (CSN3) The milk protein gene CSN3 is relevant in relation to milk production parameters and milk protein quality. The CSN3 gene encodes milk protein that is important for the structure and stability of casein micelles. The B allele of CSN3 is associated with higher milk protein content and milk quality.

6 Materials and methods The HRM assay was validated by screening genomic DNA samples of known genotypes from 488 cattle and 125 bulls. The quantitative PCR (qpcr) was performed on a LightCycler Nano Real-Time PCR System. Data was analyzed using HRM (high resolution melt analysis and genotyping) provided by the LightCycler Nano Software 1.0. DNA samples with known genotype were firstly tested by PCR-RFLP and IEF techniques. PCR-RFLP analysis: For CSN3 a 350 bp fragment was amplified and then digested with the HindIII restriction enzyme. For BLAD a 134 bp fragment was amplified and then digested with the TaqI restriction enzyme.

7 PCR-HRM Optimization Strategy PCR quality Primers annealing temperature should be ~60 C Short amplicons Purified PCR primer (HPLC) Low primer concentrations (200nM) to prevent primerdimers Pure and identical sample material Samples extraction with an identical extraction method The same DNA amount and identical buffer solution Every reaction contains the same amount (20 ng) of template DNA in the same reaction volume (20 μl) Signal reach the plateau phase

8 Optimize MgCl2 Concentration Amplification curves Effect of increasing MgCl2 on amplification behavior The concentration of Mg +2 supplied in the PCR buffers is normally not enough to ensure efficient amplification of template. An increase in the Mg +2 concentration to 2-3 mm significantly enhances the amplification. Increased Mg +2 concentration enhance nonspecific amplification and therefore has to be optimized for each primer set.

9 qpcr-hrm CSN3 BLAD Step 1: Amplification Step 2: Normalization Step 3: Difference plot

10 Normalized temperature-shifted melting curves produced by the HRM analysis CSN3 BLAD HRM analysis allows clear discrimination between different genotypes of CSN3 and BLAD. All samples were with known genotypes and were grouped correctly by the LightCycler Nano Software.

11 Difference curve Screening genomic DNA samples of known genotypes for the CSN3 and BLAD validated the assay. The difference plots in the HRM assay clearly separate samples according to their genotype, which are easily distinguished based on shape of the melting curves. Discrimination of different genetic variants is based on differences in melting temperature (Tm).

12 HRM vs. PCR-RFLP Step 1: DNA isolation Step 1: DNA isolation Step 2: DNA measurement Step 2: DNA measurement Step 3: PCR Step 3: PCR Step 4: HRM Step 4: RFLP Step 5: Electrophoresis

13 Conclusions We genotyped variants of CSN3 and BLAD occurring in the investigated population and shown that HRM is a closed tube, homogenous genotyping assay, applicable for fast detection of genetic variants in cattle. HRM analysis characterizes samples by their dissociation behavior, which is based on sequence length, GC content and DNA sequence complementarity, and can be used to detect single base sequence variations. When HRM genotyping accuracy was compared to PCR-RFLP and IEF assay no discordant results were observed. The commonly used PCR RFLP technique was laborious and time-consuming comparing with qpcr- HRM where we obtained the results faster and with less labor. High-resolution melting analysis is convenient because no processing or separation steps are required. In addition to genotyping, high-resolution melting analysis is an accurate mutation scanning tool. In summary, our results show that the HRM technique is a simple, rapid, reliable and cost-effective assay that allows genotyping of cattle for BLAD and CSN3 locus, based on post-pcr melting curve analysis under high-resolution conditions. Acknowledgements This work was published during the project Postdoctoral School of Agriculture and Veterinary Medicine", POSDRU/89/1.5/S/62371

14 Thank you for your attention!