Kidd Lab 

DECTECTION USING FLUORESCENT POLARIZATION

1. Polymerase Chain Reaction (PCR)

Template: Genomic DNA 100-200 ng/reaction volume of 10 µl

Primer 1.     NCAM1TAF:          5'-CCC AGA GCC AGA CAT TCT TAA-3'                  100 ng/reaction
Primer 2.     NCAM1FPR :         5'-CGA GTT AGG GAA ATT AGA GCT GAT-3'                    100 ng/reaction

dNTPs: Final Concentration 200 µM each

Buffer: Final Concentration 0.9 mM MgCl₂, 20 mM KCl, 4 mM Tris-HCl pH 8.4

Taq DNA Polymerase: 0.125 Units/reaction
                                 (Perkin-Elmer Amplitaq DNA polymerase 5 U/µl, Cat. No. N808-0145)

PCR Cycling Profile: 95^oC (5')
                              95^oC (15"), 65^oC (15"), 72^oC (15")  × 40 cycles
                              72^oC (10')

Product size:
                    48 bp
 
 

2. Primer and dNTP Degradation

Template: product from above per reaction

Buffer: Final Concentration 5 mM MgCl₂, 10 mM Tris-HCl pH 8.0

Shrimp Alkaline phosphatase: Final concentration 0.05 Units/ul

ExonucleaseI: Final concentration 0.05 Units/ul

PCR Cycling Profile: 37°C (90')
                              95^oC (15')

3. Single Base Extension(T/A SNP)

Template: product from SAP/Exo reaction

Primer 1.     NCAM1TAF:          5'-CCC AGA GCC AGA CAT TCT TAA-3'

ddNTPs: Final Concentration 25 µM each

Buffer: Final Concentration 6.5 mM MgCl₂, 26 mM Tris-HCl pH 9.5

ThermoSequenase: 0.43Units/reaction volumn of 30 ul
                             (USB ThermoSequenase DNA Polymerase 4 U/ul , Cat. No. 78500)

Terminator Base Extension Cycling Profile: 95°C (1')
                                                               95^oC (10"), 55^oC (30"),   × 20 cycles

Reference:
Chen, X., Lev ine, L., and Kwok, P.Y. 1999. Fluoresence polarization in homogeneous nucleic acid analysis. Genome Research 9: 492-498.
 
 

Kidd Lab Histograms for Allele Frequencies