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Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

Wheat stripe rust caused by Puccinia striiformis f.sp. tritici (Pst) is one of the most important diseases of wheat worldwide. Detection of latent infection in leaves is critical for estimating the initial inoculum potential of epidemics. A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Pst DNA in spores and wheat seedlings with latent infections. LAMP assay could detect as low as 2 pg/μl template DNA and detect latent infections from leaves as early as 24 h after inoculation. This provides a rapid and accurate method of estimating latent infection levels.

Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.

Background Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.