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Applications in Genetics
Table 1. Primer sequences and hybridization probes for detection of five CF mutations
CFTR (GenBank Acce~sion #HSACOOOJ J I)
Position
Length GC(%) Tm (DC)
R347P primers
CAGCCTTCTTCTTCTCAGGGT
79860
21
52.4
66.96
TTGTTTATTGCTCCAAGA
80046R
18
33.3
55.23
Product
79860-80046
187
R347P probes
TGACCGCCATG~GT* AGAAC-F
79988R
19
57.9
64.54
LCRed640-TGCAGAATGAGATGGTGGTG
79967R
33
42.4
70.88
AATATTTTCCGGA-P
£\F508 primers
GTGATTTGATAATGACCTAAIAATGATGG
99129
29
31
63.25
GCTTTGATGACGCTTCTGTATCTATATTC
99346R
29
37.9
65.94
Product
99129-99346
218
F508 probes'
CAGTTTTCCTGGATTAIGCCTGGCACCATT-F 99284
30
46.7
73.06
LCRed705-AGAAAATATCATCJIIGGTGTTT
99316
30
30
65.61
CCTATGA-P
G542XJG55lD/R553X primers
ATATGATTACATTAGAAGGAAGATGTGCC
127357
29
34.5
67.16
TTTACAIGAATGACATTTACAGCAAATG
127596R
28
28.6
65.70
Product
127357-127596 240
G542X probes'
GAITCCACCTTCTC,CAAGAACTAT-F
127499R
24
41.7
65.78
LCRed705-TGTCTTTCTCTGCAAACTTGG
127473R
38
39.5
74.53
AGATGTCCTATTACCAA-P
G55lD/R553X probes'
GTGGAG~TC~GAGCAAGAA-F
127507
21
52.4
68.59
LCRed640-TCTTTAGCAAGGTGAATAACT
127530
39
33.3
7\.36
AATTATTGGTCTAGCAAG-P
.. A deliberately introduced mismatch in the R347P detection probe.
I Variable nucleotides in the detection probes are underlined.
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Melting curve
Segment 1
Segment 2
Segment 3
Target temperature [DCl
95
40
75
Incubation time [sJ
10
120
0
Temperature transition rate [OC/s)
20
20
O.L
Acquisition Mode
None
None
Cont.
Gains
F1=li F2=IOi F3=80