Detection of a Single Base Substitution in Single Cells by Melting Peak Analysis •
Table 1. Oligonucleotides
Oligonucleotides (5'.3') for 2841G-,/T mutation in BReA J
Position
Length
GC(%) Tm (0C)
GCT CCG ITT TCA AAT CCA G
2727
19
47.4
66.7
CAGITTCGITGCCTCCTCTGAAC
2990
20
50.0
67.0
Product
283
40.6
82.2
Hybridization probes
AGAAACAAAGTCCAAAAGTCACITITGAAT·F
2797
30
30.0
72.6
Wtld type: LCRed640·GAACAAAAGGAAGAA·
2829
25
32.0
69.3
AATCAAGGAA·p
Mutant: LCRed705-GAACAAAAGGAAT AAA2829
25
21.8
67.3
ATCAAGGAA-p
Oligonucleotides (5'·3') for 35 I 9G-,/'[ mutation in BReAI
GGCCAAAATTGAATGCTATG
3349
20
40.0
66.5
ITGCAGTCAAGTCTTCCAAT
4025
20
40.0
66.4
Product
696
39.8
83.0
Hybridization probes (sense strand):
Anchor: CTGTTAAIACAGATTTCTCTCCA·
3475
31
32.3
68.9
TATCTGAT·F
Wild type: LCRed640·TCAGATAACITAGA·
3507
25
40.0
66.4
ACAGCCTATGG·p
Mutant: LCRed705·TCAGATAACTTATAAC·
3507
25
36.0
64.3
AGCCTATGG·p
peR reaction mix for 3519G~T mutation in BRCA1:
Volume lId]
IFinal]
FastStart DNA Master Hybridization Mix
2
Ix
MgClz (20 mM)
2.4
4mM
BRCA1 primers (5 IJM)
1+1
0.251JM
Anchor probe (2 IlM)
I
0.1 IlM
Wild· type probe (2 IlM)
0.75
75nM
Mutant probe (2 IJM)
0.75
75nM
Template (I cell)
2
HPLC grade water
9.1