PIiI Applications in Genetics
Sample Preparation We investigated a boy with CPSI deficiency who had died at the age of 28 days
of severe hyperammonemia. We checked his gene by sequencing after PCR and
identified an 840G>C mutation in one allele, which led to a 9-pb deletion at
nucleotide 832-840 in the cDNA as a result of aberrant splicing (GenBank
Accession #YI5793). The same mutation was reported previously [7]. For this
trial we prepared three templates: the RT products of the total RNAs isolated
from the liver of the patient (having the 9-bp deletion heterozygously) and of a
patient with ornithine transcarbamoylase (OTC) deficiency (as a control), and
a plasmid (pGEM-T easy plasmid) containing the homozygous 9-bp deletion
mutation. Total RNAs from the patients' livers were prepared using the modified
acid guanidinium-phenol-chloroform method [8]. First-strand cDNAs were
synthesized in an 8-f.LI mixture containing 1 f.Lg total RNA, 240 U M-MLV reverse
transcriptase, 4 f.LI 5 X reverse transcription buffer, 0.5 f.Lg random primers,
0.25 mM dNTP, and 30 U RNase-inhibitor. The reaction was performed at 37°C
for 1 h.
Oligonucleotides For fluorescence PCR analysis, we prepared two PCR primers (see Table 1).
lighteycler peR The following master mix was used for the reaction:
LightCycIer-DNA Master SYBR Green I
MgCI2 stock solution
Primers (10 IJ-M)
H 2 0 (PCR grade)
Total volume
Table 1. Oligonucleotides
Volume I~II
[Final]
2.0
2.4
0.4+0.4
12.8
18.0
Ix
4mM
0.2~M
Detection of 2-bp deletion in the GLA gene (GenBank Accession #XI4448)
Posit jon
Length GC (%)
Primers
GGGCCACTTATCACTAGTTGC
10928
21
52.4
TGATGAAGCAGGCAGGAT
1I114R
18
50.0
Product
10928- 11114 187
Probes
GTGGGAACGACCTCTCTCAG-F
10995
20
50.0
LCRed640-CTTAGCCTGGGCTGTAGCTATGATA-P
11016
25
48.0
Detection of9-bp deletion in the CPSI gene (GenBank Accession #Y15793)
Primers
GCAGAACCACTAATTCAG
GCTCCTTGCGATCACTCT
Product
811
865R
811 - 865
18
L8
55
44.4
55.6
Tm (0C)
70.8
64.5
72.3
74.2
62.2
66.8
Sample Preparation We investigated a boy with CPSI deficiency who had died at the age of 28 days
of severe hyperammonemia. We checked his gene by sequencing after PCR and
identified an 840G>C mutation in one allele, which led to a 9-pb deletion at
nucleotide 832-840 in the cDNA as a result of aberrant splicing (GenBank
Accession #YI5793). The same mutation was reported previously [7]. For this
trial we prepared three templates: the RT products of the total RNAs isolated
from the liver of the patient (having the 9-bp deletion heterozygously) and of a
patient with ornithine transcarbamoylase (OTC) deficiency (as a control), and
a plasmid (pGEM-T easy plasmid) containing the homozygous 9-bp deletion
mutation. Total RNAs from the patients' livers were prepared using the modified
acid guanidinium-phenol-chloroform method [8]. First-strand cDNAs were
synthesized in an 8-f.LI mixture containing 1 f.Lg total RNA, 240 U M-MLV reverse
transcriptase, 4 f.LI 5 X reverse transcription buffer, 0.5 f.Lg random primers,
0.25 mM dNTP, and 30 U RNase-inhibitor. The reaction was performed at 37°C
for 1 h.
Oligonucleotides For fluorescence PCR analysis, we prepared two PCR primers (see Table 1).
lighteycler peR The following master mix was used for the reaction:
LightCycIer-DNA Master SYBR Green I
MgCI2 stock solution
Primers (10 IJ-M)
H 2 0 (PCR grade)
Total volume
Table 1. Oligonucleotides
Volume I~II
[Final]
2.0
2.4
0.4+0.4
12.8
18.0
Ix
4mM
0.2~M
Detection of 2-bp deletion in the GLA gene (GenBank Accession #XI4448)
Posit jon
Length GC (%)
Primers
GGGCCACTTATCACTAGTTGC
10928
21
52.4
TGATGAAGCAGGCAGGAT
1I114R
18
50.0
Product
10928- 11114 187
Probes
GTGGGAACGACCTCTCTCAG-F
10995
20
50.0
LCRed640-CTTAGCCTGGGCTGTAGCTATGATA-P
11016
25
48.0
Detection of9-bp deletion in the CPSI gene (GenBank Accession #Y15793)
Primers
GCAGAACCACTAATTCAG
GCTCCTTGCGATCACTCT
Product
811
865R
811 - 865
18
L8
55
44.4
55.6
Tm (0C)
70.8
64.5
72.3
74.2
62.2
66.8