n Applications in Genetics
M
2 3
a
55
'5
U
i J
r:
15
05
0
-45
I
45
b
r--O
4 5 M
I
55
. - 218 bp
. - 187bp
I
I
fill
Ii6
,_rtI
O~ . ~
5
3
4
2
Fig. la-f. Agarose gel electrophoresis and melting peak analysis of PCR products from four different DNA samples and a negative control. The assignment of the samples to the melting peaks
and the lanes in the agarose gel are shown by numbers. The result of the multiplex PCR for CF
mutation loci R347P and M508 is shown in a-c. PCR of a region of the CFTR gene encompassing CF mutations G542X, G551D, and R553X is shown in d-f. Melting curves were converted to
melting peaks by plotting the negative derivative of the fluorescence with respect to temperature
(-dF/dT) against temperature. The melting point analyses of probes labelled with LCRed640
(probes for detection of R347P and G551D/R553X, respectively) are shown in band e. The melting point analyses of probes labelled with LCRed705 (probes for detection of M508 and G542X,
respectively) are shown in e and f. PCR products removed from glass capillaries by centrifugation
were separated on 3% agarose gels. M, 100 bp molecular weight marker. 1, negative control (H20);
2, R347P and M508 heterozygous; G542X, G55ID, and R553X wild type; 3, G542X and M508 heterozygous; R347P, G55ID, and R553X wild type; 4, G551D heterozygous; R347P, M508, G542X,
and R553X wild type; 5, R553X heterozygous; R347P, M508, G542X, and G551D wild type