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
Sample #
Forward
Reverse 1 G C 2 3 4 5
G/A
C/T 6 7 8 9 10 11 12

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 Sequence Multiplex Limited Throughput Medium
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.
Sequence
Multiplex
Limited
Throughput
Medium

12. SNaPshot Methodology Your Lab: DNA Analysis Facility
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 Sequence SNaPshot Multiplex Limited Yes, 7-10 SNP’s
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.
Sequence
SNaPshot
Multiplex
Limited
Yes, 7-10 SNP’s
Throughput
Medium

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.
Sequence
SNaPshot
ADA
Multiplex
Limited
Yes,
7-10 SNP’s No
Throughput
Medium
High

28. DHPLC Methodolgy DNA Analysis Facility Run sample on Transgenomic Wave
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. AR SNP was unable to be interpreted.
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.
Sequence
SNaPshot
ADA
DHPLC
Muliplex
Limited
Yes,
7-10 SNP’s No
Throughput
Medium
High

34. SNPlex Methodology

35. SNP’s: Which Method is Best for Your Study?
How many SNP’s?
How many samples?
Sequence
SNaPshot
ADA
DHPLC
SNPlex
Multiplex
Limited
Yes,
7-10 SNP’s No
48 SNP’s
Throughput
Medium
High

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.