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SNP’s: Which Method is Best for Your Study?

SNP’s: Which Method is Best for Your Study?. SNP Detection Methods available in the DNA Analysis Facility. Sequence SNaPshot Allelic Discrimination Assay DHLPC SNPlex. Joint RG Study.

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SNP’s: Which Method is Best for Your Study?

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  1. SNP’s: Which Method is Best for Your Study?

  2. SNP Detection Methods available in the DNA Analysis Facility • Sequence • SNaPshot • Allelic Discrimination Assay • DHLPC • SNPlex

  3. Joint RG Study Unfortunately, little information is available regarding the specific advantages and limitations of even the most routinely used mutation detection techniques. The primary goal is to compare operational parameters for the most popular mutation/polymorphism screening methods. In the first year of this joint proposal, we will conduct a pilot study engaging only members of the participating research groups: DSRG, FARG and NARG. This pilot study will allow us to refine the survey design and prepare a broader based study for year 2 directed at the greater scientific community.

  4. Sequencing Methodology Your Lab: • gDNA extraction • PCR amplification of region of interest DNA Analysis Facility Cycle Sequence Reaction Sequence Run

  5. Sequencing: Data Analysis TYMS SNP: AAGTTTTTACACTTT(C/T)ATTTCTCTGTGGCT

  6. Sequencing: Data Analysis Observed Mixed Ratios of Heterozygous peaks

  7. Sequence Data Analysis Confirmed Mixed Ratios of Heterozygous peaks

  8. Joint RG Data: Sequencing TNF GAGGGGCATG(A/G)GGACGG

  9. Joint RG Data: Sequencing AR SNP: Forward sequence slips on CAG repeat

  10. Joint RG Study Sequence Data Comparison TYMS, MTHFR, and TNF SNP’s • 32/36 calls matched the true sample identity. Of the four calls missed: • 3 calls missed the 2nd allele when it was at 5%. • 1 call missed the 2nd allele when it was at 12.5%. AR SNP • 12/12 calls matched the true sample identity in Reverse direction only.

  11. Sequence Summary Advantages: well established technology, easy sample preparation, and can easily get confirmation from 2nd strand. Disadvantages: Some problems with sequence context, only limited ability to multiplex SNP’s.

  12. SNaPshot Methodology Your Lab: • gDNA extraction • PCR amplification • Purify PCR product • SNaPshot Reaction • SAP treatment DNA Analysis Facility • Add size standard and perform Genescan Run

  13. SNaPshot Analysis Liz 120 Size Standard

  14. Joint RG Data: SNapShot

  15. Joint RG Study SNaPshot Data Comparison TYMS, MTHFR, TNF and AR SNP’s • 44/48 calls matched the true sample identity. Of the 4 calls missed: • 3 calls missed the 2nd allele when it was at 5%. • 1 call missed the 2nd allele when it was at 12.5%.

  16. SNaPshot Summary Advantages: this system is designed for multiplexing and you have two options: multiplex the SNaPshot reaction or pooled separate reactions. Disadvantages: primers need to be specifically designed, primers >30bp need to be HPLC purified, optimization of the SNaPshot reaction can take time.

  17. Real-time qPCR Method

  18. Allelic Discrimination Assay Methodology Your Lab: • gDNA extraction • PCR amplification with 2 dual-labelled probes DNA Analysis Facility • Perform End point plate read and Analysis

  19. Allelic Discrimination Assay:Data Analysis

  20. Joint RG Data

  21. Joint RG Data

  22. Joint RG Data

  23. Joint RG Data

  24. Joint RG Data

  25. Joint RG Data

  26. Joint RG Study ADA Data Comparison TYMS and MTHFR (both alleles represented) • 19/24 calls matched the true sample identity. Of the 5 calls that were missed: • 2 calls missed the 2nd allele when it was at 5%. • 2 calls missed the 2nd allele when it was at 12.5%. • 1 call missed the 2nd allele when it was at 25%. TNF and AR SNP’s (no 2nd allele present) • 16/24 calls matched the true sample identity. • 8 heterozygous samples were miscalled as homozygous.

  27. Allelic Discrimination Assay:Summary Advantages: ABI has a huge collection of pre-designed SNP assays so little optimization is needed. Disadvantages: can’t multiplex.

  28. DHPLC Methodolgy Your Lab • gDNA extraction • Design specific primers • PCR amplification DNA Analysis Facility • Run sample on Transgenomic Wave

  29. DHPLC Data Analysis

  30. Joint RG Data MTHFR SNP

  31. Joint RG Data AR SNP was unable to be interpreted.

  32. Joint RG Study DHPLC Data Comparison TYMS and MTHFR SNP’s • 19/24 calls matched the true sample identity. Of the 5 missed calls: • 2 calls missed the 2nd allele when it was at 5%. • 2 calls missed the 2nd allele when it was at 12.5% • 1 sample was a low signal. AR and TNF SNP’s • AR: all calls undetermined • TNF: couldn’t design suitable primers

  33. DHPLC Summary Advantages: high throughput screening method Disadvantages: can only distinguish heterozygous from homozygous with no ability to make actual base calls, some sequence parameters can make primer design difficult.

  34. SNPlex Methodology

  35. SNP’s: Which Method is Best for Your Study? How many SNP’s? How many samples?

  36. DNA Analysis Facility The facility provides: • Consultations on assay selection. • Comprehensive support for assay design. • Fast and inexpensive sample processing. • Comprehensive support for data analysis.

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