Assay

Validation status definitions:

Validated: A SNP assay has been designed and optimized for this target, and the results are concordant with another assay, (e.g. sequencing results for the SNP500Cancer 102 population, or genotyping results for the HapMap population).
Not Validated: A SNP assay has been designed and optimized for this target, however the result of this assay is the only genotyping result for this SNP, and therefore there is nothing to compare the results to validate the assay.
No Variation: A SNP assay has been designed and optimized for this target, however this assay does not exhibit variation in the SNP500Cancer 102 population, and cannot be validated.

Order number not available. Primers and probes must be ordered individually and formulated as below, or contact ABI for more information

	Concentration	5' dye	Sequence	3' dye	Allele
Probe 1	200 nM	FAM	ACGCCTGTAATCC	MGB	T
Probe 2	200 nM	VIC	CACGCCTGTCATCC	MGB	G
Primer F	900 nM		CTCGTGATCTGCCCACCTT
Primer R	900 nM		CCCCCCAAAAAATCTCTGGATATCG

    PCR Program Name: TFVMGB60
Step	Temperature/Action 	Time
1	50 deg C		2:00 minutes
2	95 deg C		10:00 minutes
3	92 deg C		0:30 seconds
4	60 deg C		1:00 minute
5	Goto Step 3		49 times
6	4 deg C			Hold

Method: 5 ng of sample DNA is dried down and used to do a 5 uL Taqman (5' nuclease assay) reaction. Reactions are set up using 2.5 ul of the 2X Universal Master Mix (Applied Biosystems, Foster City, CA) and assay-specific concentrations of primers and probes (above). All reactions are set up in a 384 (96*4) well plate and heat-sealed using an ABgene ALPS 300 heat sealer and clear heat sealing film (ABgene, Rochester, NY). Reaction plates are thermocycled as above, and endpoint reads are conducted on the ABI 7900HT sequence detection system. Cluster Analysis is conducted on the scatter plot of Allele 1 Rn versus Allele 2 Rn. Genotypic segregation is displayed in the allelic plot, containing four distinct clusters, which represent the NTCs (no template controls) and three possible genotypes clusters along the horizontal, vertical and diagonal axes, which represent the Allele 1, Allele 2 and Allele 1/Allele 2 respectively. The data are then exported in text format for further analysis.

For assay ordering information from Applied Biosystems (Foster City, CA), please note the assay number and ABI Order number (above), which can be used to reference this assay at Applied Biosystems.