Abstract
Universal or whole genome amplification by polymerase chain reaction (PCR) is a rapid and efficient method to generate fragments representing the target sequence, as well as to increase a limited amount of template. One of the most common PCR protocols for total genome amplification is the interspersed repetitive sequence-PCR (IRS-PCR) in which primers specific for human repeat-rich regions are used to generate PCR products between adjacent repeated sequences (1). However, although IRS-PCR across regions such as Alu families of human repeat has been demonstrated to be useful, the nonuniform distribution of repeat-rich region within the human genome has been a limitation. Alternative strategies have been proposed. In the primer-extension preamplification (PEP), multiple rounds of extensions with Taq DNA polymerase and a random mixture of 15-base oligonucleotides as primers produce multiple copies of the template present in the sample (2-5). In a more demanding protocol, called linker adaptor-PCR, RsaI restricted genomic DNA fragments are ligated to SmaI-cut pUC plasmid. Subsequently, the inserts are amplified by PCR using the universal M13/pUC sequencing and reverse sequencing primers and then released by EcoRI digestion (6). The tagged random primer PCR (T-PCR) is a two-step PCR strategy which consists of a pool of all possible 3′-sequences for binding to the target DNA and a constant 5′-region for the detection of incorporated primers (7). Recently, degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was developed to allow random amplification of DNA from any source (8-10). DOP-PCR uses a partially degenerate sequence in a PCR protocol with two different annealing temperatures. It has been successfully applied for amplifying entire genomes such as human, mouse, and fruit fly, as well as isolated human chromosomes and cosmids (11). The technique has also been used to prepare whole chromosome paint probes (11 12 for micro-FISH assays (13-15), comparative genomic hybridization (16), to increase the amount of sample for genotyping (17), and genomic fingerprinting (18). The DOP-PCR primer consists of three regions. The 5′-end carries a recognition sequence for XhoI (C·TCGAG), a restriction endonuclease that cuts rarely within the human genome. This sequence can be used for cloning, if desired. The sequence is then followed by a middle portion containing six nucleotides of degenerate sequence (NNNNNN, where N = A, C, G, or T in approximately equal proportions) and a 3′-end sequence containing six specific bases (ATGTGG) which primes the reaction approximately every 4 kb (8 9). The principle of the technique is that at a sufficiently low annealing temperature only the six specific nucleotides included in the 3′-end of the degenerate oligonucleotide will anneal to the genomic strand allowing the primer to initiate PCR. The PCR fragments are then generated which contain the full length of the oligoprimer at one end and its complementary sequence at the other end. Subsequently, the temperature is increased to the level required for the full length of the degenerate primer to anneal. For additional details, we direct the reader to the original papers (8 9.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nelson, D. L., Ledbetter, S. A., Corbo, L, Victoria, M. F., Ramirez-Solis, R., Webster, T. D., et al. (1989) Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA 86, 6686–6690.
Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidi. W., and Arnheim, N. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl. Acad. Sci. USA 89, 5847–5851.
Snabes, M. C., Chong, S. S., Subramanian, S. B., Kristjansson, K., DiSepio D., and Hughes, M. R. (1994) Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification. Proc. Natl. Acad. Sci. USA 89, 6181–6185.
Barrett, M. T., Reid, B. J., and Joslyn, G. (1995) Genotypic analysis of multiple loci in somatic cells by whole genome amplification. Nucleic Acids Res. 23, 3488–3492.
Barrett, M. T., Galipeau, P. C., Sanchez, C. A., Emond, M. J., and Reid, B. J. (1996) Determination of the frequency of loss of heterozygosity in esophageal adenocarcinoma by cell sorting, whole genome amplification and microsatellite polymorphisms. Oncogene 12, 1873–1878.
Ludecke, H. J., Senger, G., Claussen, U., and Horsthemke, B. (1989) Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338, 348–350.
Grothues, D., Cantor, C. R., and Smith, C. L. (1993) PCR amplification of megabase DNA with tagged random primers (T-PCR). Nucleic Acids Res. 21, 1321–1322.
Telenius, H., Carter, N. P., Bebb, C. E., Nordenskjold, M., Ponder, B. A. J., and Tunnacliffe, A. (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13, 718–725.
Telenius, H., Pelmear, A. H., Tunnacliffe, A., Carter, N. P., Behmel, A, Ferguson-Smith, M. A., et al. (1992) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes, Chromosomes & Cancer 4, 257–263.
Speicher, M. R., du Manoir, S., Schrock, E., Holtgreve-Grez, H., Schoell, B., Lengauer, C., et al. (1993) Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumor by comparative genomic hybridization after universal DNA-amplification. Hum. Mol. Genet. 2, 1907–1914.
Scalzi, J. M. and Hozier, J. C. (1998) Comparative genome mapping: mouse and rat homologies revealed by fluorescence in situ hybridization. Genomics 47, 44–51.
Burkin, D. J., O’Brien, P. C., Broad, T. E., Hill, D. F., Jones, C. A., Wienberg, J., and Ferguson-Smith, M. A. (1997) Isolation of chromosome-specific paints from high-resolution flow karyotypes of the sheep (Ovis aries). Chromosome Res. 5, 102–108.
Engelen, J. J., Loots, W. J., Albrechts, J. C., Plomp, A. S., van der Meer, S. B., Vles, J. S., et al. (1998) Characterization of a de novo unbalanced translocation t(14q18q) using microdissection and fluorescence in situ hybridization. Am. J. Med. Genet. 75, 409–413.
Xiao, Y., Darroudi, F., Kuipers, A. G., de Jong, J. H., de Boer, P., and Natarajan, A.T. (1996) Generation of mouse chromosome painting probes by DOP-PCR amplification of microdissected meiotic chromosomes. Cytogenet. Cell. Genet. 75, 63–66.
Goureau, A., Yerle, M., Schmitz, A., Riquet, J., Milan, D., Pinton, P., et al. (1996) Human and porcine correspondence of chromosome segments using bidirectional chromosome painting. Genomics 36, 252–262
Kuukasjarvi, T., Tanner, M., Pennanen, S., Karhu, R., Visakorpi, T., and Isola, J. (1997) Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization. Genes, Chromosomes & Cancer 18, 94–101.
Cheung, V. G., and Nelson, S. F. (1996) Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc. Natl. Acad. Sci. USA 93, 14676–14679.
Sayada, C., Picard, B., Elion, J., and Krishnamoorthy, R. (1994) Genomic fingerprinting of Yersinia enterocolitica species by degenerate oligonucleotide-primed polymerase chain reaction. Electrophoresis 15, 562–565.
Cheng, J., Waters, L. C., Fortina, P., Hvichia, G., Jacobson, S. C., Ramsey, J. M., et al. (1998) Degenerate oligonucleotide primed-PCR and capillary electrophoretic analysis of human DNA on microchip-based devices. Anal. Biochem. 257, 101–106
McGillis, D. A. (1983) In: VLSI Technology. (Sze, S. M., ed.), McGraw Hill, New York, NY, pp. 267–280.
Katz, L. E. (1983) In: VLSI Technology (Sze, S. M., ed.), McGraw Hill, New York, NY, pp. 131–167.
Cheng, J., Shoffner, M. A., Hvichia, G. E., Kricka, L. J., and Wilding, P. (1996) Chip PCR, II. Investigation of different PCR amplification systems in micro-fabricated siliconglass chips. Nucleic Acids Res. 24, 380–385.
Shoffner, M. A., Cheng, J., Hvichia, G. E., Kricka, L. J., and Wilding, P. (1996) Chip PCR, I. Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Res. 24, 375–379.
Poncz, M., Solowiejzcyk, D., Harpel, B., Moroy, Y., Schwartz, E., and Surrey, S. (1982) Construction of human gene libraries from small amounts of peripheral blood: Analysis of b-like globin genes. Hemoglobin 6, 27–33.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Beggs, A. H., Koenig, M., Boyce, F. M., and Kunkel, L. M. (1990) Detection of 98% of DMA/BMD gene deletions by polymerase chain reaction. Hum. Genet. 86, 45–48.
Fortina, P., Cheng, J., Shoffner, M. A., Surrey, S., Hitchcock, W. H., Kricka, L. J., and Wilding, P. (1997) Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis. Clin. Chem. 43, 745–751.
Jacobson, S. C., Hergenröder, R., Koutny, L. B., Warmack, R. J., and Ramsey, J. M. (1994) Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices. Anal. Chem. 66, 1107–1113.
Hjertén, S. (1985) High performance electrophoresis: elimination of electroendosmosis and solute adsorption. J. Chromatogr. 347, 191–198.
Jacobson, S. C. and Ramsey, J. M. (1996) Integrated microdevice for DNA restriction fragment analysis. Anal. Chem. 68, 720–723.
Ulfelder, K. J., Schwartz, H. E., Hall, J. M., and Sunzeri, F. J. (1992) Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis. Anal. Biochem. 200, 260–267.
Waters, L. C., Jacobson, S. C., Kroutchinina, N., Khandurina, J., Foote, R. S., and Ramsey, J. M. (1998a) Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal. Chem. 70, 158–162.
Waters, L. C., Jacobson, S. C., Kroutchinina, N., Khandurina, J., Foote, R. S., and Ramsey, J. M. (1998b) Multiple sample PCR amplification and electrophoretic analysis on a microchip. Anal. Chem. 70, 5172–5176.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Fortina, P. et al. (2001). DOP-PCR Amplification of Whole Genomic DNA and Microchip-Based Capillary Electrophoresis. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology™, vol 163. Humana Press. https://doi.org/10.1385/1-59259-116-7:211
Download citation
DOI: https://doi.org/10.1385/1-59259-116-7:211
Publisher Name: Humana Press
Print ISBN: 978-0-89603-765-6
Online ISBN: 978-1-59259-116-9
eBook Packages: Springer Protocols