Presentation on theme: "Brown trout, coho, pink, and sockeye salmon populations were screened for SNPs that will enable us to differentiate component populations in mixed stock."— Presentation transcript:
Brown trout, coho, pink, and sockeye salmon populations were screened for SNPs that will enable us to differentiate component populations in mixed stock analysis. Cutthroat and rainbow trout populations were screened for SNPs which could be used in species differentiation and the idenfication of possible hybrids in areas where their ranges overlap. REFERENCES 1.Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, Radel MQ, Goldman D. DNA melting analysis for detection of single nucleotide polymorphisms. Clinical Chemistry 2001; 47:4, 635-44. 2.Herrman MG, Durtschi JD, Bromley KL, Wittwer CT, Voelkerding KV. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clinical Chemistry 2006; 52:3, 494–503. ACKNOWLEDGEMENTS We thank Molly Stephens (University of California Davis), Sewall Yong (Washington Dept. of Fish and Game), Todd Seamons (University of Washington) and Steven Kalinowski (Montana State University) for providing DNA samples. HRM result verified by TaqMan® SNP Genotyping. The assay was designed using FileBuilder ® and ordered from Applied Biosystems. TaqMan® allelic discrimination experiment was performed on 7900HTHRM result verified by TaqMan® SNP Genotyping. The assay was designed using FileBuilder ® and ordered from Applied Biosystems. TaqMan® allelic discrimination experiment was performed on 7900HT High Resolution Melting Analysis for Genotyping Cell Free Fetal DNA Ebru Dündar YENİLMEZ, Duygu DÜZGÜNCE, Abdullah TULİ Çukurova University, Faculty of Medicine, Biochemistry Department, Saricam /Balcali, 01330, Adana,TURKEY email@example.com Materials and Methods Maternal blood (5mL) was collected from 50 women at 7-12 weeks of gestation who undergo to CVS. Plasma was separated by centrifugation at 1600 g and then 16000 g within 1 hour. Fetal DNA was extracted from plasma with QIAamp blood mini kit according to manufacturer instructions. HRM analysis was used to identify HbS mutation of plasma fetal DNA. Light Cycler was used for high resolution melting assay of 50 impregnate plasmas, Hemoglobin S/C Toolset kit for LightCycler was used according to manufacturer instructions 3. Quantitative fluorescent polymerase chain reaction assay (QF- PCR) and Short Tandem Repeat (STR) markers analyzed with AMPISTR kit by manufacturers instructions to detect fetal alleles in the corresponding maternal plasma samples, when the maternal and fetal DNA was HbS carrier. In order to confirm the data of fetal DNA, CVS was analyzed by both HRM and traditional methods; restriction fragment length polymorphism (RFLP) and amplification refractory mutation system (ARMS) 5. Introduction Sickle cell anemia (SCA) is one of the most common human autosomal recessive disorders and is prevalent in the Çukurova region of southern Turkey. The incidence of sickle cell trait is 10.0% in the Çukurova region. Prenatal diagnosis of SCA has been available by conventional prenatal diagnostic procedure such as chorionic villus sampling (CVS) in our prenatal diagnosis centre since 1992. This invasive procedure carries a risk of miscarriage about 1-2%. Because of this risk, cell free fetal DNA in maternal plasma is being increasingly studied in pregnancy in recent years. The discovery of fetal DNA in maternal plasma has opened up new opportunities in non-invasive prenatal diagnosis. Detecting the mutation with cell free fetal DNA non-invasively is an important goal of prenatal diagnosis. High Resolution Melting analysis (HRM) combined with real- time PCR was introduced in 1997. It is a new method for DNA analysis especially for genotyping and mutation scanning. Fluorescence analysis of DNA melting is more sensitive than invasive methods and only nanogram amounts of sample are needed. Aim of The Study The aim of this study was detection of SCA mutation (HbS) by HRM analysis in fetal DNA from maternal plasma. Results In our study, we used HRM analysis to detect HbS in fetal DNA as an alternative to invasive techniques (ARMS, RFLP). Wild type control and homozygous patients presented a clearly distinguishable difference in Tm (56 and 63°C, respectively). Samples which were analyzed heterozygous for HbS, showed Tm peaks values between wild type and the mutant samples (Figure A, B and C). In 28 fetal samples with heterozygous HbS, the parents did not share the same mutations so that the paternal mutation in maternal plasma would indicate the presence of paternal mutation in the fetus. When the fetus carries the same genotype as the mother it was difficult to seperate fetal and maternal DNA. We used STR analysis to see different loci that comes from the father. A. Mother is beta thalassemia trait, father is SCA trait (AS) and the fetus observed heterozygous for SCA (AS). B. Mother is beta thalassemia trait (AA), father is SCA trait (AS) and the fetus observed heterozygous for SCA (AS). C. Homozygous wild type (AA) control and the fetus observed homozygous mutant for (SS). Conclusion Sickle cell anemia is a severe, lifelong disease that can be accurately diagnosed at prenatal period by invasive methods such as CVS. Because the procedure carries a risk of miscarriage the development of new non-invasive techniques using fetal DNA and fetal cells in maternal circulation has been initiated. The scarce amount of fetal DNA within total DNA in maternal circulation makes non-invasive prenatal diagnosis for single gene disorders difficult. In our study, we used HRM analysis combined with real-time PCR, which can be operated with slight amount of DNA, to diagnose sickle cell mutation prenatally at fetus. The HRM results were confirmed by traditional techniques (RFLP, ARMS) at CVS samples. The samples which carried the same genotype with mother were analyzed by STR. In 72% of the samples, paternally inherited fetal alleles detected by STR were found to be informative and identification of loci different from mother helped the differentiation of fetus from mother. HRM analysis combined with real-time PCR has become an alternative method to classical PCR procedures because of its being more practical, easier to perform, cost effective and less time- consuming. Detecting SCA at the samples using CVS gives result with 100% accuracy by HRM analysis. If maternal fetal DNA carries same mutation with mother, HRM analysis cannot always discriminate between fetus and mother. In order to obtain absolute results with fetal DNA either usage of different STR markers specific to the disease studied or designation of more specific probes are suggested. Acknowledgements This project was funded by Çukurova University Science Foundation Grant (TF2005D2). References 1.Lo YMD, Tein MSC, Lau TK et al Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am. J of Human Genet. 1998; 62: 768-775. 2.Marziliano N, Pele E, Minuti B et al. Melting temperature assay for a UGT1A gene variant in Gilbert Syndrome. Clin.Chem. 2003; 46(6): 853-60. 3. Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid b-globin genotyping by multiplexing probe melting temperature and color. Clin.Chem. 2000;46:425-28. 4.Wittwer CT, Reed GH, Gundri CN et al. High resolution genotyping by amplicon melting analysis using LCGreen. Clin.Chem. 2003; 46(6): 853-60. 5.Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res, 1989; 17:2503-2516. GenotypeFetus Hb AA – wild type12 Hb AS – heterozygous28 Hb SS – mutant10 Heterozygous (AS) father Heterozygous (AS) fetus Mother (AA) Heterozygous (AS) fetus Homozygous mutant fetus(SS) Homozygous (AA) wild type In our study, we tried to use HRM analysis with real- time PCR to genotype the fetus from plasma DNA for HbS. Table1 shows the samples we established as wild type, heterozygous and mutant in fetal DNA by HRM analysis. Table 1. Sample genotypes by HRM analysis.