A. Fawad Faruqi

Comprehensive human genome amplification using multiple displacement amplification

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.

Triple-Helix Formation Induces Recombination in Mammalian Cells via a Nucleotide Excision Repair-Dependent Pathway

ABSTRACT The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of asupF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Targeted Correction of an Episomal Gene in Mammalian Cells by a Short DNA Fragment Tethered to a Triplex-forming Oligonucleotide

Triplex-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner and provoke DNA repair. We have coupled a TFO to a short donor fragment of DNA that shares homology to a selected gene as a strategy to mediate gene targeting and correction. In this bifunctional oligonucleotide, the TFO domain is designed to bind the target gene and stimulate repair and recombination, with the donor domain positioned for recombination and information transfer. A series of these tethered donor-TFO (TD-TFO) molecules with donor domains of 40–44 nucleotides and TFO domains in both the purine and pyrimidine triplex motifs were tested for their ability to mediate either gene correction or mutation of a supF reporter gene contained in a SV40 shuttle vector in mammalian cells. In vitro binding assays revealed that the attachment of the donor domain via a flexible linker did not significantly alter the binding affinity of the TFO domain for the polypurine site in the supF target DNA, with equilibrium dissociation constants in the 10−8 m range. Experiments in which the target vector and the linked TD-TFOs were pre-incubated in vitro and co-transfected into cells led to conversion frequencies approaching 1%, 4-fold greater than with the two domains unlinked. When cells that had been previously transfected with the SV40 vector were electroporated with the TD-TFOs, frequencies of base pair-specific gene correction were seen in the range of 0.04%, up to 50-fold over background and at least 3-fold over either domain alone or in unlinked combinations. Sequence conversion by the TD-TFOs was achieved using either single- or double-stranded donor domains and either triplex motif. Substitution of either domain in the TD-TFO with control sequences yielded reagents with diminished activity, as did mixtures of unlinked TFO and donor DNA segments. The boost in activity provided by the attached TFO domain was reduced in cells deficient in the nucleotide excision repair factor XPA but was restored in a subclone of these cells expressing XPA cDNA, suggesting a role for nucleotide excision repair in the pathway of triple helix-stimulated gene conversion. The ability to correct or mutate a specific target site in mammalian cells using the TD-TFO strategy may provide a useful tool for research and possibly for therapeutic applications.

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

BackgroundRolling 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.ResultsA 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.ConclusionsCombining 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.