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assay to distinguish the three alleles is based on
the 309bp-long fragment that encompasses one
SNP for the
mo-1
2
allele, and one SNP and the
deletion for the
mo-1
1
allele (Figure 14.4). To
increase resolution, this assay uses an unlabeled
probe that overlaps the single SNP differentiat-
ing
Mo-1
and
mo-1
2
alleles.
Asymmetric PCR amplification was per-
formed in a 10 μl reaction volume containing
10 ng of genomic DNA as a template, 1
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PCR
buffer, 1 mM MgCl
2
,0.75U
Ta q
polymerase, 0.2
mM of each dNTP (all from New England Bio-
labs, Ipswich, MA), 1
×
LCGreen Plus Melting
Dye (Idaho Technology, Salt Lake City, UT), 0.1
μM of forward primer (Mo-1-F: 5'- GTA CGG
CCA TAG CTC AGC A -3'), 0.5 μMofreverse
primer (Mo-1-R: 5'- GCA ACG TAT ACA GCC
AAC AGG -3'), and 0.5 μM of probe (Mo-1-
probe: 5'- CGA TAC TCC CTC TCC TAA GTC
CAA GC /3SpC3/ -3' with a carbon spacer at the
3' end to block extension on that end). The reac-
tion was filled to the final volume with nuclease-
free water; 15 μl of mineral oil was added to
prevent evaporation during HRM analysis. PCR
was performed with an initial denaturation of 95
◦
C for 2 min, followed by 55 cycles of 95
◦
Cfor
30 s, 63
◦
C for 30 s, and 72
◦
C for 30 s, with
final extension of 72
◦
C for 5 min. To facilitate
heteroduplex formation, samples were heated to
95
◦
C for 30 s, followed by cooling to 25
◦
C
for 30s. Melting profiles of the three alleles are
shown in Figure 14.3 (bottom row).
×
Acknowledgements
The author would like to thank Drs. M. Jeuken,
J. McCreight, R. Hayes, and B. Mou for review-
ing the manuscript; A. Atallah for editing the
manuscript; and A. Atallah, D. Pechenick, L. Lai,
and M. Chawner for technical assistance with
designing and running MAS assays. Confiden-
tial information provided by colleagues from the
private sector is greatly appreciated. Develop-
ment of assays for marker-assisted selection was
supported in part by the California Leafy Greens
Research Program. Mention of trade names or
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