A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

Development of microdissection and chromosome specific genomic library in Lilium tigrinum

Techniques for microdissection and microcloning were established using chromosome 1 of triploid Lilium tigrinum. Chromosome 1 was dissected from a membrane slide using a microbeam system. Digestion with proteinase K was done before PCR amplification for more than 24 hours. The dissected chromosomes were then amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and linker adaptor-mediated PCR (LA-PCR). Successful PCR amplification relied on critical concentrations of both MgCl₂ and Taq polymerase. The optimum concentration of MgCl₂ and Taq polymerase was 2.5 mM and 0.5 U, respectively. Amplification of the dissected chromosome using DOP-PCR had more sensitivity dependent upon PCR factors, but LA-PCR was more dependent on the linker ligation. Amplified DNA products ranged from 100 to 2500 bp both for DOP- and LA-PCR. Evaluated clones only ranged from 100 to 1700 bp for DOP-PCR and 100 to 900 bp for LA-PCR. Based on the sequence results, most of the sequences from the DOP-PCR and LA-PCR showed no significant similarity with known data in NCBI database. However, about 2% of the sequence data was partially matched with plant microsatellites with low similarity. The results derived from the microdissection of a large genome organism such as Lilium showed informative and useful for the development of microsatellite repeat markers. Sequence data from the chromosome specific DNA library was considered for the development of microsatellite markers.     

Comparison of Amplification Methods of Single Trophoblast Cells for Their Subsequent Analysis Using Metaphase Comparative Genomic Hybridization

Single trophoblast cells circulating in the bloodstream of pregnant women are potential objects for noninvasive prenatal diagnosis. Owing to the very low concentration of cells of a fetal nature in the peripheral maternal blood, the choice of the method for whole genome amplification of the genetic material becomes topical. The key point in the use of single cells of a fetal nature for noninvasive prenatal diagnosis is to obtain DNA in an amount and of a quality acceptable for the analysis. In order to select the optimal method for whole genome amplification, a model experiment was conducted. We compared three different methods of whole genome amplification: linker-adaptor polymerase chain reaction (LA-PCR), degenerate oligonucleotide- primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Subsequent analysis of the amplification products was performed by metaphase comparative genomic hybridization in order to evaluate the molecular karyotype of cells of a fetal nature with the known chromosome complement. As a result, an optimal method for whole genome amplification of the genetic material of single cells in a model experiment was determined by linker-adaptor PCR, which showed a more uniform representation of the genome regions compared with the other methods used.     

Long DOP-PCR of rare archival anthropological samples

The application of molecular DNA technologies to anthropological questions has meant that rare or archival samples of human remains, including blood, hair, and bone, can now be used as a source of material for genetic analysis. Often, these samples are irreplaceable, and/or yield very small quantities of DNA, so methods for preamplifying as much of the whole genome as possible would greatly enhance their usefulness. DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction) is an amplification method that uses a degenerate primer and very low initial annealing temperatures to amplify the whole genome. We adapted a published DOP-PCR protocol to long PCR enzyme and amplification conditions. The effectiveness of these modifications was tested by PCR amplification of DOP-PCR products at a mixture of genomic targets including 66 different microsatellites, 11 Alu insertion polymorphisms, and variable-length segments of the human lipoprotein lipase gene (LPL). The selected microsatellite markers were chosen to represent every chromosome, with expected product sizes ranging from 150 base pairs to 8,000 base pairs in length, while the 22 Alu insertion polymorphisms were selected to reveal biases in the recovery of alleles of different sizes. To determine nucleotide sequence variation, 2 kilobases (kb) of the LPL gene in 30 Mongolian individuals were sequenced. All gene-specific targets from DOP-PCR product template were amplified. No unexpected polymorphisms in the sequence results attributable to the DOP-PCR step were found, and 93% to 95% of Alu genotypes that have been amplified from total genomic DNA were replicated. The incorrect typings were all due to the preferential amplification of the shorter of two possible alleles in individuals heterozygous for an Alu insertion and were all correctly typed on subsequent reamplification of the gene-specific PCR products. This method of whole-genome amplification promises to be an efficient way to maximize the genetic use of rare anthropological samples.     

Optimization of DOP-PCR amplification of DNA for high-resolution comparative genomic hybridization analysis

BACKGROUND: Whole-genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. METHODS: Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and fluorescence labeling were applied to test DNA from normal and K-562 cells. The DNA products were used for CGH analysis. RESULTS: The DOP-PCR-amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal-to-noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP-PCR-protocol was further investigated. We observed concordance between CGH results using unamplified and DOP-PCR-amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. CONCLUSIONS: DOP-PCR-amplified DNA is applicable for high- resolution CGH, the results being similar to those of CGH using unamplified DNA.     

Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii

Background

Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls.

Results

All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR.

Conclusion

Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.     

High resolution comparative genomic hybridization detects 7-8 megabasepair deletion in PCR amplified DNA.

We investigated if any change in spatial resolution of comparative genomic hybridization analysis could be detected when using DNA amplified by degenerate oligonucleotide primed PCR (DOP-PCR) as opposed to the use of unamplified DNA. Five DNA samples from B-cell leukemias with small 11q deletions were amplified by DOP-PCR and analysed by means of high resolution comparative genomic hybridization (HR-CGH) for the evaluation of aberration size detection limit. By means of HR-CGH, we found the detection limit of DOP-PCR CGH for deletions to be between 3 Mbp and 7-8 Mbp.     

A Whole Genome Amplification Method to Generate Long Fragments from Low Quantities of Genomic DNA

Several whole genome amplification strategies have been developed to preamplify the entire genome from minimal amounts of DNA for subsequent molecular genetic analysis. However, none of these techniques has proven to amplify long products from very low (nanogram or picogram) quantities of genomic DNA. Here we report a new whole genome amplification protocol using a degenerate primer (DOP-PCR) that generates products up to about 10 kb in length from less than 1 ng genomic template DNA. This new protocol (LL-DOP-PCR) allows in the subsequent PCR the specific amplification, with high fidelity, of DNA fragments that are more than 1 kb in length. LL-DOP-PCR provides significantly better coverage for microsatellites and unique sequences in comparison to a conventional DOP-PCR method.     

Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.

A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.     

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

A PCR-based amplification method retaining the quantitative difference between two complex genomes

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

Heterogeneous chromosomal aberrations in intraductal breast lesions adjacent to invasive carcinoma.

There is evidence that breast cancer is a heterogeneous disease phenotypically as well as molecular biologically. So far, heterogeneity on the molecular biological level has not been investigated in potential precursor lesions, such as ductal hyperplasia (DH) and ductal carcinoma in situ (DCIS). In this study we applied comparative genomic hybridization (CGH) to formalin-fixed, paraffin-embedded breast tissue with DH and DCIS, adjacent to invasive ductal carcinoma (IDC), to screen these potential precursor lesions for whole genomic chromosomal imbalances. Laser-microdissection was used to select pure cell populations from the sections. Isolated DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and further processed for CGH analysis. Investigating multiple samples (n = 25) from four patients we found an average of 5.6 +/- 0.9 (mean +/- SEM) chromosomal imbalances already present in DH. In the twelve DCIS lesions an average of 10.8 (+/- 0.9) aberrations was identified with 14.8 (+/- 0.8) aberrations in the four adjacent IDC lesions. The increasing number of chromosomal changes in parallel with the histopathological sequence corroborate the hypothesis, that the carcinomas may have developed through a sequential progression from normal to proliferative epithelium and eventually into carcinoma. However, heterogeneous results were identified in the multiple samples per entity from the same patient, demonstrated mainly in the DCIS samples in the chromosomal regions 6p, 9p, 11q, 16p and 17q, in the DH samples by 3p, 16p and 17q. This heterogeneous findings were most pronounced within the DH and was less in the DCIS and IDC samples. The only aberration consistently found in all samples-even in all DH sample-was amplification of the 20q13 region. Our results demonstrate, that the applied combination of laser-microdissection, DOP-PCR and CGH, may serve to analyse breast carcinogenesis pathways in suitable histological material. However, so far, it is unclear how to handle heterogeneous results and these make identification of relevant changes more difficult. Setting a threshold and evaluating only those chromosomal changes which are present in a majority of samples may be one possibility. This involves however, the risk that infrequent but possibly significant aberrations may be missed. Figures on http://www.esacp.org/acp/2000/20-1/aubele. htm.     

Single-primer PCR correction: a strategy for false-positive exclusion

Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single- and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.     

SNP genotyping on a genome-wide amplified DOP-PCR template

With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/µl improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Quantification of trace-level DNA by real-time whole genome amplification

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.     

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.