n Methods Useful in Genetics and Oncology
6p21.3-p21.1 [2]. A 150-bp segment of intron 1 is amplified in standard DNA and
in test DNA in the same conditions. This amplification precisely reflects the number of genomic copies present in the sample.
Materials
LightCycler instrument and software (Roche Diagnostics, Mannheim, Germany)
LightCycler capillaries, centrifuge adaptors and cooling blocks (Roche Diagnostics)
Human Blood (from the lab staff)
Genomic DNA (Clontech, Palo Alto, USA)
LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics)
LightCycler FastStart DNA Master Hybridization Probes (Roche Diagnostics)
QIAGEN Genomic Tips (100/G) (Qiagen, Hilden, Germany)
QIAGEN Genomic DNA Buffer Set
QIAGEN Protease
Retinoic X Receptor B primers and probes designed, synthesized, and purified
(HPLC) by TIB MOLBIOL (Berlin, Germany)
Procedure
Sample Preparation Human genomic DNA was isolated from 5 ml of heparinized blood. Mononuclear
cells were isolated by isopycnic centrifugation through Ficoll-Histopaque (Sigma
St Louis, USA) and processed according to QIAGEN genomic DNA purification
Handbook. DNA was resuspended in 10 mM Tris, pH 8. DNA concentration was
estimated by the ethidium bromide fluorescence technique and DNA quality was
evaluated with ultra violet absorbance. Stock sample DNA solutions (approximately 500-100 ng/Ill) were diluted to 1:10 and 1:100.
A dilution series in the range of 100-0.01 ng/fll of Clontech genomic DNA was
used for external standardization. Since the mass of one human haploid genome
is 3 pg, these dilutions correspond approximately to 33,333-3.3 copies of the single-copy RXRB gene.
Oligonucleotides are shown in Table 1.
Table 1. Oligonucleotides
Homo sapiens Retinoic X Receptor B gene (GcnBank Accession ItAF065396)
Position Length GC(%) Tm (0C)
PCR primers
GAG CGA CGG GCT TAA TIC GA
7L4
20
55
60.3
GAG CGG CCC AAG ACA TG
864
17
64.7
57.5
PCR Product
150
56.7
86.7
Hybridization probes
TCG GAG GAT TAG CTG AGC ACG AGG A-X
792
25
56.0
65.7
LCRed640-CCC CTG AGA GAA AGA erc TGG cer G-P 765
25
60.0
66.2
6p21.3-p21.1 [2]. A 150-bp segment of intron 1 is amplified in standard DNA and
in test DNA in the same conditions. This amplification precisely reflects the number of genomic copies present in the sample.
Materials
LightCycler instrument and software (Roche Diagnostics, Mannheim, Germany)
LightCycler capillaries, centrifuge adaptors and cooling blocks (Roche Diagnostics)
Human Blood (from the lab staff)
Genomic DNA (Clontech, Palo Alto, USA)
LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics)
LightCycler FastStart DNA Master Hybridization Probes (Roche Diagnostics)
QIAGEN Genomic Tips (100/G) (Qiagen, Hilden, Germany)
QIAGEN Genomic DNA Buffer Set
QIAGEN Protease
Retinoic X Receptor B primers and probes designed, synthesized, and purified
(HPLC) by TIB MOLBIOL (Berlin, Germany)
Procedure
Sample Preparation Human genomic DNA was isolated from 5 ml of heparinized blood. Mononuclear
cells were isolated by isopycnic centrifugation through Ficoll-Histopaque (Sigma
St Louis, USA) and processed according to QIAGEN genomic DNA purification
Handbook. DNA was resuspended in 10 mM Tris, pH 8. DNA concentration was
estimated by the ethidium bromide fluorescence technique and DNA quality was
evaluated with ultra violet absorbance. Stock sample DNA solutions (approximately 500-100 ng/Ill) were diluted to 1:10 and 1:100.
A dilution series in the range of 100-0.01 ng/fll of Clontech genomic DNA was
used for external standardization. Since the mass of one human haploid genome
is 3 pg, these dilutions correspond approximately to 33,333-3.3 copies of the single-copy RXRB gene.
Oligonucleotides are shown in Table 1.
Table 1. Oligonucleotides
Homo sapiens Retinoic X Receptor B gene (GcnBank Accession ItAF065396)
Position Length GC(%) Tm (0C)
PCR primers
GAG CGA CGG GCT TAA TIC GA
7L4
20
55
60.3
GAG CGG CCC AAG ACA TG
864
17
64.7
57.5
PCR Product
150
56.7
86.7
Hybridization probes
TCG GAG GAT TAG CTG AGC ACG AGG A-X
792
25
56.0
65.7
LCRed640-CCC CTG AGA GAA AGA erc TGG cer G-P 765
25
60.0
66.2