Robust Dosage PCR (RD‐PCR) for Highly Accurate Dosage Analysis

Vu Q. Nguyen^1,2, Qiang Liu¹, Steve S. Sommer^1,2

¹ City of Hope National Medical Center and Beckman Research Institute, Duarte, California, ² MEDomics, Azusa, California

Publication Name: Current Protocols in Human Genetics

Unit Number: Unit 9.20

DOI: 10.1002/0471142905.hg0920s60

Online Posting Date: January, 2009

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Abstract

Clinical diagnostic and epidemiological assays would benefit from accurate detection of duplications and deletions commonly missed by conventional methods of polymerase chain reaction (PCR) amplification and sequencing of individual exons. Robust dosage-PCR (RD-PCR) is a quantitative PCR method that co-amplifies a target template and an endogenous internal control (an autosomal and an X-chromosomal segment) for detection of these mutations. RD-PCR has the advantage of high accuracy and consistency, rapid assay development, widely available controls, and gene dosage over a wide dynamic range. Curr. Protoc. Hum. Genet. 60:9.20.1-9.20.12. © 2009 by John Wiley & Sons, Inc.

Keywords: ratio of yield (ROY); ratio of template (ROT); GC content; gene dosage

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Introduction
Basic Protocol 1: Treatment of Genomic DNA by Proteinase K Followed by Heat Incubation
Basic Protocol 2: Primer Design
Basic Protocol 3: Determining Copy Number
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Treatment of Genomic DNA by Proteinase K Followed by Heat Incubation

Materials

Assay solution (see recipe)
1 mg/ml calf thymus DNA standard solution (see recipe)
100% ethanol
Genomic DNA, isolated from blood (see appendix 3B) in TE buffer, pH 8.0 (appendix 2D)
TE buffer (pH 8) 20 mg/ml proteinase K (Roche)
TE buffer, pH 8.0

Hoefer DyNA Quant 200 fluorometer (Hoefer)
4-ml glass cuvette (Hoefer)
Thermal cycler

Basic Protocol 3: Determining Copy Number

Materials

RD-PCR reaction master mixes (see Table 9.20.2)
Forward and reverse oligonucleotide primers for target (see Basic Protocol 2 for design) and control (Table 9.20.2) loci
30 ng/µl deproteinated genomic DNA in TE buffer, pH 8.0 (Basic Protocol 1)
Agarose
1× TBE buffer (appendix 2D)
6× gel loading buffer (see recipe)
10 mg/ml ethidium bromide (Sigma)

PCR tubes (appropriate for thermal cycler)
Automated thermal cycler
10-cm gel tray (minimum length)
Gel comb for 30-µl wells
UV-based gel documentation system
Laser-based gel imaging system (Typhoon 9410 Variable Mode Imager; GE Healthcare Life Science)
Image analysis software (ImageQuant; GE Healthcare Life Science)

Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)

Table 9.20.2 Master Mixes for the Three RD-PCR Conditions

			Assay master mixes^b (µl)

Component	Final concentration	Per reaction^a	I	II	III

10× Expand High Fidelity Buffer #3 (Roche)	1×	2.5 µl	87.5	87.5	87.5
5 mM 4dNTP mix (1.25 mM each; Roche)^c	200 µM each	4.0 µl	140.0	140.0	–
6.25× dNTP/dGTP/deaza-dGTP mix^c,d	1×^d	4.0 µl	–	–	140.0
Control forward/reverse primer mix (2.5 µM each)	0.1-0.4 µM	1.0 to 4.0 µl	V^e	V^e	V^e
Target forward/reverse primer mix (2.5 µM each)	0.1-0.4 µM	1.0 to 4.0 µl	V^e	V^e	V^e
50 mM MgCl₂ (I and II)^f	4.5 mM	2.25 µl	78.75	78.75	–
50 mM MgCl₂ (III)^f	3.0 mM	1.5 µl	–	–	52.5
5 M betaine	1.0 mM	5.0 µl	–	–	175.0
0.5 µg/µl BSA (Roche)	0.02 µg/µl	1.0 µl	35.0	35.0	35.0
5 U/µl Platinum High Fidelity Taq Polymerase (Invitrogen)	0.04 U/µl	0.2 µl	7.0	7.0	7.0
5 U/µl Platinum Taq Polymerase (Invitrogen)	0.04 U/µl	0.2 µl	7.0	7.0	7.0
H₂O	V^g	4.85 µl	169.75	169.75	169.75

^aThe assay master mix per tube is 15 µl. The final reaction volume per tube is 25 µl.

^bTotal volume = 525 µl. Number of reactions = 35 (enough for n + 1 reactions).

^cMake the dNTP mixes, dispense into 500 µl aliquots, and store up to 1 year at –20°C.

^ddATP, dCTP and, dTTP at 1.25 mM each; dGTP at 0.9375 mM; deaza-dGTP at 0.3125 mM (Roche). Final concentrations are 200 µM dNTP/150 µM dGTP/50 µM deaza-dNTP. Deaza-dGTP partially substitutes for dGTP and reduces the number of hydrogen bonds with complementary dCTP. This prevents formation of large stable DNA complexes.

^eVariable: volume to be determined via optimization of ratio of target primer mix to control primer mix.

^fMgCl₂ concentration for assays I and II is different from that of assay III.

^gVariable: volume to be determined by final volumes of target and control primers.

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Figures

Figure 9.20.1 RD-PCR schematics. Target and endogenous internal control are co-amplified by RD-PCR, and the PCR products are separated by agarose electrophoresis. In the example, a family with hemophilia B is tested for a deletion of exon H in the FIX gene. The relative product ratio of target to control (ROY) is obtained for each sample. The template copy number of exon H is determined per cell from its ROY. Lanes 1 and 2 are wild-type male and female samples, respectively. Lane 3 is a female member without the deletion, lane 4 is a female carrier with 1 copy per cell, lane 5 is the male proband with a deletion, and lane 6 represents a mosaic deletion with 1.5 copies per cell. Reprinted from Analytical Biochemistry, vol. 371, Nguyen, V.Q., Liu, Q., and Sommer, S.S., A large-scale validation of dosage analysis by robust dosage-polymerase chain reaction. pp. 37-42. Copyright 2007, with permission from Elsevier.

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Literature Cited

Literature Cited
	Armour, J.A., Sismani, C., Patsalis, P.C., and Cross, G. 2000. Measurement of locus copy number by hybridisation with amplifiable probes. Nucleic Acids Res. 28:605-609.
	Cooper, D.N. and Krawczak, M. 1993. Human Gene Mutation. BIOS Scientific Publishers Limited, Oxford.
	Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Genome Res. 167:1-8.
	Ketterling, R.P., Vielhaber, E.L., Lind, T.J., Thorland, E.C., and Sommer, S.S. 1994. The rates and patterns of deletions in the human factor IX gene. Am. J. Hum. Genet. 54:201-213.
	Liu, Q. and Sommer, S.S. 1998. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: Application to the inversion hotspot in the factor VIII gene. BioTechniques 25:1022-1028.
	Liu, Q., Li, X., Chen, J.S., and Sommer, S.S. 2003. Robust dosage-PCR for detection of heterozygous chromosomal deletions. BioTechniques 34:558-570.
	Nguyen, V.Q., Shi, J., Liu, Q., and Sommer, S.S. 2004. Robust dosage (RD)-PCR protocol for the detection of heterozygous deletions. BioTechniques 37:360-364.
	Nguyen, V.Q., Liu, Q., and Sommer, S.S. 2007. A large-scale validation of dosage analysis by robust dosage-polymerase chain reaction. Anal. Biochem. 371:37-42.
	Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwinjnenburg, D., Diepvens, F., and Pals, G. 2002. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 30:e57.
	Shi, J., Liu, Q., Nguyen, V.Q., and Sommer, S.S. 2004. Elimination of locus-specific inter-individual variation in quantitative PCR. BioTechniques 37:934-938.
	Shuber, A.P., Grondin, V.J., and Klinger, K.W. 1995. A simplified procedure for developing multiplex PCRs. Genome Res. 5:488-493.
	Telenti, A., Imboden, P., and Germann, D. 1992. Competitive polymerase chain reaction using an internal standard: Application to the quantitation of viral DNA. J. Virol. Met. 39:259-268.
	Wetmur, J.G. 1991. DNA probes: Applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26:227-259.
	Wilke, K., Duman, B., and Horst, J. 2000. Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR. Hum. Mutat. 16:431-436.

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