n Methods Useful in Genetics and Oncology
Procedure
Sample Preparation
The DNA was extracted from peripheral blood leukocytes using standard phenolchloroform DNA extraction and ethanol precipitation [Thomas, 1989] and stored
in TE' (lOmM Tris, pH 8.0, O.lmM EDTA) at -20C. After extraction, the purified
genomic DNA was boiled for S minutes in a water bath. DNA from 9 individuals
with Huntington's disease and 18 normal individuals from prior studies [Bernard,
1999; Wittwer, 1993] was analyzed.
Primer Design The amplification primers extend into the CAG repeat region several base pairs
on both ends. They exclude the adjacent polymorphic CCG trinucleotide region
as suggested previously [Andrew, 1994; Rubenstein, 1993].
Oligonucleotides Table 1. Oligonucleotides
lightCycier PCR
IT-IS (I-ID) Gene, Exon 1 (Genbank Accession #NM_00211 1)
Position
Length
GC(%)
Tm (0G)
Primers
cgagtccctcaagtccttccagca
348
24
58.3
57.4
GTGGCGGCTGTTGCTGCT
442R
18
66.7
58.6
Product
348-442
56-179
The following master mix was used:
Volume IIJlI
IFinal1
dNTP's (RMB, 2mM each)
1.00
0.20 mM each
MgCl1buffer (10 mM)
1.00
1.0 mM
DMSO (100%)
0.70
7.0%
KlenTaq polymerase (diluted to 0.50U/ul)
1.0
0.05 units/l
Primers (5.0 M each)
l+1
0.50 M each
SYBR Green I (diluted 3000:1)
1
1:30,000
H20 (peR grade)
2.3
Total volume
9
9 ~ of master mix and 1~ of DNA (IO-SOng/I) were added to each capillary. The
capillaries were spun briefly and placed into the LightCycler carousel.