Soybean Genomics and Improvement : Single Base Extension

with Luminex Flow Cytometry Detection

Uses the single base extension or mini-sequencing approach for SNP detection

'Yes'/'No' detection: Labeled Terminator present vs. absent

Detection of heterozygotes requires two separate single base extension assays

Produce PCR products containing the SNP and remove excess primers and nucleotides

Denature and anneal primers in the presence of ddNTPs (one with Biotin label) and thermosequenase

Create Biotin-Streptavidin Conjugate

'de-Multiplex' using Luminex Polystyrene Beads (with Anti-ZIP Code oligos)

Two laser excitation

One laser excites the two dyes present in the internally color-coded microspheres

Second laser excites the Biotin:Streptavidin conjugate

Detection (5,000 microspheres/sec.)

Photo-avalanche tubes detect each color-coded microsphere by the ratio of the two dyes

Photomultiplier tube detects Biotin:Streptavidin emission (presence vs. absence)