Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA - PubMed (original) (raw)

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA

V G Cheung et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6-40 ng with amplifications of 200- to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.

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Figures

Figure 1

Figure 1

Genotypic analysis of genomic DNA as compared with DOP-PCR-amplified genomic DNA. The individual is homozygous at two loci, D3S2418 (97 bp, 97 bp) and D6S1006 (200 bp, 200 bp) and heterozygous at two loci, D4S2366 (127 bp, 136 bp) and D1S2130 (252 bp, 256 bp). (A) An electropherogram of a multiplex PCR at these four microsatellite loci using 40 ng of genomic DNA as template. (B) An electropherogram of a multiplex PCR at the same four loci using 40 ng of DOP-PCR-amplified genomic DNA as template. Comparison of A and B shows matching genotypes and corresponding peak areas from the two templates. The_y_-axis represents arbitrary fluorescent units, and the_x_-axis represents the size of fragments in base pairs.

Figure 2

Figure 2

Comparison of yield from the DOP-PCR product generated from serially diluted genomic DNAs. All panels show electropherograms from genotyping of mixtures of DNA with fluorescently labeled D8S1132 primers as discussed. (A) An electropherogram from a mixture of total genomic DNA from two heterozygous individuals: 40 nanograms of genomic DNA from individual 1 (allele sizes 152 bp and 162 bp) were mixed with 80 ng of genomic DNA from individual 2 (allele sizes 158 bp and 168 bp). (B_–_H) Electropherograms at D8S1132 on 40 ng of DOP-PCR product derived from a DOP-PCR of a serial dilution of starting genomic DNA of individual 1 mixed with 80 ng of genomic DNA from individual 2, which allow measurement of relative yield. DOP-PCR product used in_B_–H are derived from starting genomic DNA amounts of 400 ng, 80 ng, 16 ng, 3.2 ng, 640 pg, 128 pg, and 26 pg, respectively. Comparison of the peak areas on the electropherograms reflects how the DOP-PCR products amplify relative to genomic DNA. These data were used to determine the corrected yield in Table 1. The_y_-axis represents arbitrary fluorescent units, and the_x_-axis represents fragment size in base pairs.

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References

    1. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Proc Natl Acad Sci USA. 1992;89:5847–5851. - PMC - PubMed
    1. Snabes M C, Chong S S, Subramanian S B, Kristjansson K, DiSepio D, Hughes M R. Proc Natl Acad Sci USA. 1994;91:6181–6185. - PMC - PubMed
    1. Barrett M T, Reid B J, Joslyn G. Nucleic Acids Res. 1995;23:3488–3492. - PMC - PubMed
    1. Grothues D, Cantor C R, Smith C L. Nucleic Acids Res. 1993;21:1321–1322. - PMC - PubMed
    1. Ludecke H J, Senger G, Claussen U, Horsthemke B. Nature (London) 1989;338:348–350. - PubMed

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