J. Aquiles Sanchez

Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

ETS Protein–Dependent Accessibility Changes at the Immunoglobulin μ Heavy Chain Enhancer

Directed accessibility mediated by antigen-receptor gene enhancers ensures developmental stage-specific activation of V(D)J recombination. Here, we used a combination of in vitro and in vivo assays to explore the mechanisms that regulate immunoglobulin mu heavy chain gene enhancer-dependent chromatin accessibility. Ets-1 or PU.1 bound to mu enhancer-containing plasmids assembled into chromatin in vitro and increased restriction enzyme access to a proximal site. In complementary analyses, expression of PU.1 in Ets-1-containing 2017 pro-T cells or NIH 3T3 cells induced sterile I mu transcripts at the IgH locus and increased accessibility of the endogenous mu enhancer to restriction endonucleases. These observations suggest that one role of PU.1 is to increase accessibility of the mu locus to initiate heavy chain gene expression.

New insights into the mechanisms of nuclear segmentation in human neutrophils

During human neutrophil differentiation, large portions of the genome condense and associate with the nuclear envelope to form filament‐like structures. As a result, the nucleus of the mature neutrophil typically consists of a linear array of three or four lobes joined by thin, DNA‐containing filaments. Despite the medical significance of neutrophil nuclear morphology, little is known about the events regulating neutrophil nuclear differentiation and its pathological states. This work presents a new model of the mechanisms governing nuclear filament formation in human neutrophils. This model is based on recent chromosome mapping studies in human neutrophils and on studies of genetic and pathological conditions affecting neutrophil nuclear shape. According to this model, filament assembly is initiated by factors that interact with specific regions of the genome in a hierarchical and dose‐dependent manner. In this regard, the strategies governing the molecular interactions responsible for filament formation appear to resemble those involved in transcriptional silencing, a phenomenon that also affects the properties of extended chromosomal regions. According to the silencing paradigm, bound filament control Factors must recruit additional Filament Foehn factors which spread along adjacent DNA to mediate filament formation. A better understanding of the factors that shape the neutrophil nucleus may lead to new clinical tools for the diagnosis and manipulation of abnormal neutrophil differentiation. J. Cell. Biochem. 73:1–10, 1999.

Two-temperature LATE-PCR endpoint genotyping

BackgroundIn conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.ResultsTwo-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles.ConclusionSNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Fluorescent in situ hybridization (FISH) analysis of the relationship between chromosome location and nuclear morphology in human neutrophils

Abstract.Human neutrophil nuclei typically consist of three of four large heterochromatic lobes joined by thin, DNA-containing filaments. In addition, some lobes exhibit appendages of various sizes and shapes. Classical genetic and cytological studies suggest that some appendages contain specific chromsomes. The studies reported here provide the first detailed analysis of the spatial relationship between individual chromosomes and recognizable structures in neutrophil nuclei using fluorescent in situ hybridization. Analysis of DNA sequences in chromosomes 2, 18, X, and Y demonstrate that specific lobes in a population of neutrophil nuclei do not have a fied chromosome content. This result implies that chromosomes partition randomly among lobes during neutrophil differentiation. However, neutrophil nuclear topography is not entirely fortuitous. For instance, none of the sequences probed in this study mapped to a filament and most centromeres lie in clusters near the nuclear periphery. In addition, one of the X chromosome centromeres in females and the Y chromosome centromere in males consistently associate with specific nuclear appendages found in a subset of neutrophil nuclei. Chromosomes 2 and 18 occupy discrete nd separate territories within individual lobes and neither territory ever extends into a filament. Surprisingly, the sizes of these territories are not proportional to chromosome length, suggesting that individual neutrophil chromosomes vary in their degree of compaction. These results are discussed in the light of models that attempt to explain nuclear morphology in terms of chromosome spatial organization.

Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals.

Two-Dimensional DNA Gel Electrophoresis as a Method for Analysis of Eukaryotic Genome Structure: Evaluation Using Tetrahymena thermophila DNA

There is growing interest in mapping and analyzing complete eukaryotic genomes. Yee and Inouye (in Experimental Manipulation of Gene Expression, pp. 279-290, Academic Press, New York) demonstrated that bacterial chromosomes can be resolved into interpretable patterns of DNA fragments by means of restriction enzyme digestion and electrophoresis in two dimensions. We have begun to explore applications of this procedure to analysis of eukaryotic genomes, which are far more complex. Tetrahymena thermophila was selected as a model organism because its genome is small, roughly equivalent to that of a single human chromosome. In addition, each Tetrahymena cell contains two nuclei which differ in sequence composition and methylation. Our results demonstrate that the Tetrahymena genome can be resolved into complex patterns of fragments in two dimensions. Hybridization to Southern blots of these gels with a multiply repeated sequence probe yielded analyzable patterns of a subset of the genome. The blots reveal alterations in genome structure due to methylation and rearrangement. Future extensions of the method are discussed.

Oxidative Damage is not a Major Contributor to AZT-Induced Mitochondrial Mutations

Addition of clinically-relevant levels of 3′-Azido-3′-deoxythymidine (AZT) to cultured HepG2 cells increases the number of reactive radical species (reactive oxygen and nitrogen species [ROS and RNS]) as well as random mutations in mitochondrial DNA (mtDNA). Co-treatment of AZT-exposed cells with palm fruit juice (PFJ) mitigates AZT mutagenesis. These findings suggest that AZT-dependent mtDNA damage resulted from increased reactive species and that PFJ, a known anti-oxidant, mitigated such damage by decreasing the levels of these species. The present report tests the predictions that (1) PFJ mitigates AZT mutagenesis by reducing the burden of AZT-induced reactive species, and (2) AZT-induced mutations in mtDNA should predominantly consist of G → T and C → A substitutions characteristic of DNA oxidative damage. Levels of reactive species and mitochondrial mutagenesis were measured in HepG2 cells exposed AZT in the presence or absence of PFJ. Controls experiments showed that PFJ in HepG2 cells exhibited strong scavenging activity against hydrogen peroxide-induced ROS, the main reactive species generated by dysfunctional mitochondria. Despite this strong antioxidant activity PFJ did not decrease AZT-induced reactive species at a concentration that mitigated mtDNA mutations. Consistent with this observation, the spectrum of AZT-induced mutations did not fit the spectrum expected from direct mtDNA oxidative damage. Instead, the spectrum obtained was consistent with the majority of mutations (80%) arising from mitochondrial DNA polymerase errors induced by AZT. These observations suggest that oxidative damage was not the major contributor to AZT-induced mutations.

AZT Treatment Increases mtDNA Mutations in HepG2 and CCD-1112Sk Cells

Mitochondrial dysfunction is linked to disease, but it remains unclear whether accumulation of random mutations in the mitochondrial genome is the cause of dysfunction. Using digital or near-digital LATE-PCR with Lights-On/Lights- Off probes we have measured the mutational load in mitochondrial genomes. Exposure of HepG2 and CCD-1112Sk cells to AZT for thirty days caused a significant increase in mutations in the three mitochondrial loci examined. These results demonstrate the utility of our method for analysis of mutational load without sequencing and reinforce the fact that mitochondrial DNA damage due to drugs, aging, and disease should be studied in detail.

Single-Tube Mutation Scanning of the Epidermal Growth Factor Receptor Gene using Multiplex LATE-PCR and Lights-On/Lights-Off Probes

BACKGROUND Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21. METHODS The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation. RESULTS The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies. CONCLUSION The LATE-PCR EGFR assay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies.