F

Sample prep for 310/3100

Add GS120 LIZ size standard

ExoSAP Digestion

SNP Extension (cycle sequencing)

SAP treatment

Run on ABI 310/3100

Use E5 filter (5-dye) and POP4 standard conditions

Data Analysis (GeneScan)

Type Sample (Genotyper or GeneMapper/D)

Figure 8.1

Steps in allele-specific primer extension SNP detection (e.g., mini-sequencing or SNaPshot) assay. The boxed portions illustrate additional steps performed in this assay relative to STR typing.

competing side reactions occur and thus interfere with the desired primer extension of the SNP primer.

Following the SNP extension reaction, the products are treated with SAP to remove unincorporated fluorescent ddNTPs. If the SAP-treatment is incomplete, then dye artifacts (a.k.a. dye blobs) may occur in the electropherogram and obscure the SNP allele peaks being measured.

The availability of 5-dye detection with electrophoretic platforms (see Chapter 13) enables an internal size standard to be added in the fifth dye channel to correct for migration differences from run-to-run (see Chapter 15). Each of the four nucleotides has their own dye color: A (green), G (blue), C (yellow; usually displayed as black for better visual contrast), and T (red). Thus, the presence of a blue peak in the electropherogram would indicate that a G (ddGTP) had been incorporated by the polymerase at the SNP site.

Multiple primers can be analyzed simultaneously by linking a variable number of additional nucleotides to the 5'-end of the primers so that each primer differs by several nucleotides from its neighbor. Typically a poly(T) tail is used with a 3-5 base spread between primers (Tully et al. 1996, Vallone et al. 2004) although a mixed sequence that is not complementary to any human sequences has been used successfully (Sanchez et al. 2003). Thus, primerl may contain a 5T 5'-tail, primer2 a 10T tail, primer3 a 15T tail, and primer4 a 20T tail in order to adequately resolve each locus during an electrophoretic separation (Figure 8.2).

Figure 8.2 Allele-specific primer extension results using four autosomal SNP markers on two different samples (a). SNP loci are from separate chromosomes (1, 6, 14, and 20) and therefore unlinked. Electrophoretic resolution of the SNP primer extension products occurs due to poly-T tails that are five nucleotides different from one another (b).

4000 3000 2000 1000 0

4000 3000 2000 1000 0

Sample 1

Sample 2

TT TT CT TT

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