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 oligonucleotideprimed 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 g 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.F or genomic studies, the quality and quantity of DNA samples is critical. High-throughput genetic analysis requires large amounts of template for testing, yet typically the yield of DNA from individual patient samples is limited. Forensic and paleoarcheology work also can be severely limited by DNA sample size. An important goal is to supply a sufficient amount of genomic sequence for a variety of procedures as well as longterm storage for future work and archiving of patient samples. Methods include the time-consuming process of creating of Epstein-Barr virus-transformed cell lines and whole genome amplification (WGA) by random or degenerate oligonucleotideprimed PCR (DOP-PCR) (1-3). However, PCR-based WGA methods may generate nonspecific amplification artifacts (2), give incomplete coverage of loci (4), and generate DNA less than 1 kb long (1-3) that cannot be used in many applications.Recently, a rolling circle amplification (5) method was developed for amplifying large circular DNA templates such as plasmid and bacteriophage DNA (6). Using 29 DNA polymerase and random exonuclease-resistant primers, DNA was amplified in a 30°C reaction not requiring thermal cycling. This is made possible in part by the great processivity of 29 DNA polymerase, which synthesizes DNA strands 70 kb in length (7). Here we extend the use of exonuclease-resistant primers and 29 DNA polymerase to WGA. The amplification is surprisingly uniform across the genomic target, with the relative representation of different loci differing by less than 3-fold. In contrast, PCR-based WGA methods exhibited strong amplification bias ranging fr...

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.The multiple displacement amplification (MDA) reaction uses the 29 DNA polymerase and random primers to amplify DNA templates (1-3, 5-6). Amplification from small specimens has enabled novel research approaches (reviewed in reference 7), including genetic analysis of single blastomeres for use in preimplantation diagnosis of embryos (4). A method to amplify genomic DNA from nonculturable bacteria would allow direct analysis of virtually any microbe. We demonstrate here the use of MDA to achieve several-billion-fold amplification of genomic DNA from a single bacterium. MDA could be used for a wide range of approaches for discovery of new species, population and polymorphism analysis, diagnostics, and rapid detection of pathogens.As a test case, E. coli cells (ATCC 10798; K-12 strain) were isolated (fluorescence-activated cell sorter Vantage flow cytometer [Becton Dickinson] using CellQuest and CytoCount softwares). To demonstrate proficiency in flow sorting, 180 putative cells were collected and vigorously vortexed in 10 l phosphate-buffered saline to separate cells in the event that more than one cell was obtained, and the number of CFU was determined (Fig.

Eukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across environments.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (؊sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with ؊sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic ؊sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3 end of the ؊sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) synthesizes minus-strand strong stop DNA (ϪsssDNA) by extending tRNA 3 Lys bound at the primer binding site (PBS) in the RNA genome (8,15,37). To synthesize ϪsssDNA, RT copies the U5 and R regions; the R region contains two large hairpins known as the poly(A) hairpin and the TAR hairpin (9). When this RNA is copied into DNA, hairpins that correspond to the TAR and poly(A) hairpins of the RNA can form in the nascent ϪsssDNA; the formation of these hairpins depends on the digestion of the template RNA (17). The RNase H activity of RT cleaves the RNA portion of the RNA-DNA heteroduplex during polymerization, and there is additional RNase H cleavage after ϪsssDNA synthesis is complete (8,12,18,27,33). Although it is possible for either the nascent TAR and poly(A) DNA to form hairpins that could self-prime, such self-priming events are not detected when the HIV-1 genome is copied into DNA in infected cells. Because the TAR DNA hairpin forms at the end of R, the likelihood of self-priming is higher for TAR than for the poly(A) hairpin. Instead of self-priming, the 3Ј end of the nascent DNA is efficiently transferred to the R sequence on the 3Ј end of the template RNA, where synthesis continues. This event is known as the first-strand transfer. Nucleocapsid protein (NC) has been shown to prevent synthesis of selfpri...