Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and “amplifiability” for aCGH and other downstream applications. Phenol–chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.     

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.     

Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides

The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.     

Genotyping DNA isolated from UV irradiated human bloodstains using whole genome amplification

Molecular Biology Reports - Analysis of DNA polymorphisms are the primary technique used for personal identification in forensic cases. However, DNA samples collected as evidence from crime scenes...     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

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.     

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.     

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

PCR-mediated whole genome amplification of phytoplasmas

A method was developed for genome analysis of phytoplasmas, bacterial plant pathogens that cannot be cultivated in vitro in cell-free media. The procedure includes a CsCl-bisbenzimide gradient buoyant centrifugation followed by polymerase chain reaction (PCR)-mediated whole genome amplification. The latter step involves digestion of the DNA by a restriction enzyme with an A/T-rich recognition sequence. Due to the different A/T content in the DNA of the pathogen and its plant host, the fragments originating from phytoplasma are shorter and are preferentially amplified in the PCR reaction. Products obtained were cloned and screened by dot-blot hybridization. Results showed that about 90% of recombinant clones appeared to harbor phytoplasma specific DNA inserts. Sequencing of randomly selected clones was carried out and comparison with the NCBI database confirmed the bacterial origin for the sequences, which have been assigned a putative function. The origin of the recombinant clones was further confirmed by the generation of specific amplicons from the phytoplasma-infected plant and not from the healthy control, using PCR primers devised from the sequences of the recombinant clones. This method could be used for genome-wide comparisons between phytoplasmas.     

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.     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.

     

Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR.     

Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays

Whole genome DNA microarrays were constructed and used to investigate genomic diversity in 18 Campylobacter jejuni strains from diverse sources. New algorithms were developed that dynamically determine the boundary between the conserved and variable genes. Seven hypervariable plasticity regions (PR) were identified in the genome (PR1 to PR7) containing 136 genes (50%) of the variable gene pool. When comparisons were made with the sequenced strain NCTC11168, the number of absent or divergent genes ranged from 2.6% (40 genes) to 10.2% (163) and in total 16.3% (269) of the genes were variable. PR1 contains genes important in the utilisation of alternative electron acceptors for respiration and may confer a selective advantage to strains in restricted oxygen environments. PR2, 3 and 7 contain many outer membrane and periplasmic proteins and hypothetical proteins of unknown function that might be linked to phenotypic variation and adaptation to different ecological niches. PR4, 5 and 6 contain genes involved in the production and modification of antigenic surface structures.     

Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives

Hospital tissue repositories host an invaluable supply of diseased samples with matched retrospective clinical information. In this work, a recently optimized method for extracting full-length proteins from formalin-fixed, paraffin-embedded (FFPE) tissues was evaluated on lung neuroendocrine tumor (LNET) samples collected from hospital repositories. LNETs comprise a heterogeneous spectrum of diseases, for which subtype-specific diagnostic markers are lacking. Six archival samples diagnosed as typical carcinoid (TC) or small cell lung carcinoma (SCLC) were subjected to a full-length protein extraction followed by a GeLC-MS/MS analysis, enabling the identification of over 300 distinct proteins per tumor subtype. All identified proteins were categorized through DAVID software, revealing a differential distribution of functional classes, such as those involved in RNA processing, response to oxidative stress and ion homeostasis. Moreover, using spectral counting for protein abundance estimation and beta-binomial test as statistical filter, a list of 28 differentially expressed proteins was generated and submitted to pathway analysis by means of Ingenuity Pathway Analysis software. Differential expression of chromogranin-A (more expressed in TCs) and stathmin (more expressed in SCLCs) was consistently confirmed by immunohistochemistry. Therefore, FFPE hospital archival samples can be successfully subjected to proteomic investigations aimed to biomarker discovery following a GeLC-MS/MS label-free approach.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.