New DNA amplification methods promise to improve results of genetic and gene expression analysis of archive tissue samples.
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Collections of formalin-fixed tissue are routinely maintained in medical and research institutions as an archive of histology-based diagnoses. It is estimated that there are more than 300 million paraffin tissue block samples in the United States alone, and this number is growing at a pace of about 20 million samples per year.1 Paraffin blocks have been collected and preserved for over a century, representing a historical collection of virtually every disease. Most of these samples are associated with a rich patient medical history, making them an invaluable source for the discovery and characterization of disease biomarkers.
Unfortunately, the majority of these specimens have been fixed in formaldehyde for purposes other than genetic characterization. As a result, the diagnostic and therapeutic significance of these clinical specimens has yet to be realized on a large scale, due to the quality and condition of the DNA trapped within them. Given this situation, there is increasing need for molecular methods capable of unleashing useful genetic information from archival tissue collections. This article will provide an overview of the problems inherent with formalin-fixed, paraffin-embedded (FFPE) tissues, and provide a review of some of the current methodologies available for characterizing genomic DNA from these samples.
Fixing affects DNA quality
It is well-known that tissue fixation with formalin adversely affects DNA quality, and as a result, the quality of DNA present in FFPE tissues varies drastically from sample-to-sample. There are multiple factors that contribute to DNA damage, namely fixation conditions and the length of time the sample is stored. At least one study has implicated the latter as the most important factor influencing DNA quality,1 presumably because residual formalin within some FFPE tissues continues to react with DNA during sample storage.
Formalin reacts with nucleic acids to form at least four types of modifications:2
• Formaldehyde, the active ingredient in formalin, can react with nucleic acid bases to form mono-methylol group additions (-CH2 OH). This is the most common type of damage rendered to nucleic acids through the fixation process, and it is well-known these additions can be reversed by heating nucleic acids in buffered solutions for extended periods of time.3
• N-methylol, the predominant form of formaldehyde when in solution, can undergo electrophilic attack on an amino base to form a methylene bridge between two adjacent nucleic acid bases.
• Formaldehyde treatment can generate apurinic and apyrimidinic sites via hydrolysis of the N-glycosylic bonds.
• Formaldehyde may also cause slow hydrolysis of the phosphodiester bonds leading to strand breaks.
Much of this damage is irreversible, and if the fixation process has been carried too far, DNA from some FFPE tissues may not be of sufficient quality to allow molecular characterization. The assessment of DNA quality is a crucial first step in acquiring meaningful data from FFPE tissues. Unfortunately, the DNA adducts produced by the fixation process make quality difficult to assess. For example, gel electrophoresis does not successfully predict the utility of FFPE tissue DNA for PCR, as separate strands can be cross-linked.4 Spectrophotometry, picogreen, and other commonly-used DNA characterization methods often over-estimate the amount of amplifiable DNA present in compromised samples. DNA lesions disrupt hybridization, polymerization, ligation, and other reactions used for characterization. It is imperative to establish the viable amount of nucleic acids in archival samples that are capable of participating in downstream reactions.
 click to enlarge This figure illustrates the principle and mechanism of GenomePlex. (Source: Sigma-Aldrich) |
Archived samples: PCR analysis
Real-time qPCR assays have emerged as the method of choice to measure quantities and assess quality of degraded nucleic samples. Several methods have been published demonstrating the usefulness of PCR-based assays for assessing DNA quality from FFPE tissue DNA.1,4,5,6,7,8 Multiplex PCR assays using primer sets that amplify fragments of increasing size are effective quality control tools for identifying damage, as the relationship between the ability to amplify long stretches of DNA is inversely proportional to the amount of lesions accumulated in the FFPE DNA.9 Fragmentation is also a problem with fixed samples, and attempts to PCR-amplify fragments longer than 200-bp are often unsuccessful, while fragments <120-bp are amplified success rates approaching 100%.4
There are a few products on the market designed to repair damaged DNA and improve PCR success, namely New England Biolabs’ (Ipswich, Mass.) PreCR Repair Mix and Sigma-Aldrich’s (St. Louis, Mo.) Restorase DNA Polymerase. PreCR Repair Mix is a cocktail of DNA repair enzymes designed to repair a broad range of DNA damage, including abasic sites, thymine dimers, nicks and gaps, deaminated cytosine and 8-oxo-guanine, as well as other types of lesions, prior to PCR. Restorase is a blend of Sigma’s proofreading DNA polymerase with a DNA repair enzyme that promotes repair and amplification of damaged DNA. Both products require a brief, 37°C incubation prior to PCR in order to facilitate DNA repair, and both companies caution that their products cannot rescue fragmented and other various forms of damage.
Other PCR-based technologies useful for analyzing genetic information from FFPE tissues are MRC-Holland’s (Amsterdam, The Netherlands) Multiplex Ligation-dependent Probe Amplification (MLPA) technology and Sigma-Aldrich’s Extract-N-Amp Tissue PCR Kit. MLPA is a multiplex PCR method that can detect copy number changes, amplification, and deletions in a single reaction in up to 45 specific nucleic acid sequences using a single PCR primer pair. MLPA assays are designed to allow the researcher to distinguish various chromosomal rearrangements upon separation of the amplicons by capillary electrophoresis. Amplification product length varies between 120-bp and 480-bp, allowing analysis from all but the most highly damaged FFPE DNA templates. Sigma-Aldrich’s Extract-N-Amp Tissue PCR Kit uses an all-liquid, integrated extraction and amplification system to amplify genomic DNA from FFPE and other tissues by PCR or qPCR. Tissue processing time is 15 minutes and all steps can be automated via a liquid handler.
Typical DNA yields from FFPE samples are in the sub-microgram range, which eliminates applications requiring large amounts of DNA (e.g., chromatin immunoprecipitation (ChIP), array-based comparative genomic hybridization microarrays (aCGH), SNP analysis, massively-paralleled serial sequencing (MPSS), etc.) for such samples. There are a number of commercially-available whole genome amplification (WGA) products on the market; a few have been designed with the intent of amplifying genomic DNA from FFPE tissues.
Several reports have demonstrated the utility of Sigma’s GenomePlex WGA technology for amplifying microgram quantities of FFPE DNA from as little as 10ng of template.10,11,12,13 GenomePlex is based on a modified DOP-PCR technology14 capable of amplifying fragments as small as 400-bp, but the best performance is achieved when fragments are > 1-kb in length. The GenomePlex WGA Tissue Kit amplifies genomic DNA directly from 0.1 to 1mg archival tissue via a proteinase K digestion. After protease heat-inactivation, the sample can be WGA-amplified. Multiple displacement amplification (MDA) is another popular technology for WGA of DNA from various sources.15 Several groups have applied MDA-based WGA to FFPE samples, but the requirement of long template DNA has hindered this method from being widely used with archived samples.11,16,17
Recently, Enzo Life Sciences, Farmingdale, N.Y., and Qiagen, Venlo, The Netherlands, have modified MDA to produce WGA kits specifically for amplifying genomic DNA from FFPE tissue. The Enzo Life Sciences BioScore Screening and Amplification kit discriminates FFPE DNA quality based on the yield of amplification product produced in a one-hour, isothermal WGA reaction that is capable of generating more than 10µg of DNA from 10ng high-quality template DNA. Qiagen’s approach to amplifying archival DNA via the REPLI-g FFPE Kit involves a two step-process; FFPE DNA fragments are randomly ligated together to produce high molecular weight DNA, which undergoes MDA WGA. DNA fragments <500-bp in length are not a suitable substrate for this kit, nor will the kit work if using <500 genome equivalents of template. Like Sigma’s GenomePlex WGA Tissue Kit, the REPLI-g FFPE Kit uses a one-hour proteinase K digestion step to release DNA directly from archival tissue prior to WGA.
Archived samples: array analysis
There are two enabling array-based technologies that are suitable for analysis of archival DNA samples. One such technology, Illumina’s (San Diego, Calif.) GoldenGate Assay, provides a unique solution for interrogating up to 1,536 single nucleotide polymorphism (SNP) loci from 250ng of DNA. The assay requires stretches of target DNA <40-bp on each side of the SNP of interest, making it ideal for genotyping and loss of heterozygosity (LOH) detection with damaged FFPE samples.18,19,20 In practice, the GoldenGate Assay can tolerate stretches of target DNA >200-bp, which opens this method to fixed samples. This bead array system is based on allele-specific primer extension reactions directly on genomic DNA. Three primers are designed for each SNP to be interrogated. Each primer contains a universal linker region that acts as a target site in a subsequent PCR amplification. Two of these primers are specific to each SNP allele (allele-specific oligos—ASO), and the third primer hybridizes several bases downstream of the SNP serving as a locus-specific oligo (LSO). After hybridization and extension of the proper allele specific oligo(s) and ligation of the extended product to the locus specific oligo, a subsequent PCR amplification ensues to generate enough material for array hybridization. Arrays are available for assaying either 16 or 96 samples simultaneously, requiring a total of about six hours of hands-on-time. Each array contains 50,000 beads distributed among 1,520 bead types, so that each bead type is represented at 30-fold redundancy.
Agilent Technologies, Santa Clara, Calif., has teamed up with Kreatech Biotechnologies, Amsterdam, The Netherlands, to provide an alternative for processing FFPE samples via aCGH. Their new Oligo aCGH Labeling Kit for FFPE Samples has been designed for use with Agilent’s aCGH array platforms, and is based on Kreatech’s Universal Linkage System (ULS). ULS is a non-enzymatic labeling method, directly coupling of Cy3 or Cy5 labels to DNA. The technology is independent of fragment length and requires between 0.25ng and 2µg of FFPE DNA depending on which Agilent aCGH array the material is to be hybridized.
In conclusion, formaldehyde reacts with DNA, introducing adducts or lesions that are recalcitrant to most molecular biology manipulations. While there are a number of commercially-available technologies for obtaining useful genetic information from fixed tissues, there still remains a tremendous need for enabling technologies capable of overcoming the pitfalls of working with damaged DNA. Development of new technologies capable of obtaining useful data from the smallest fragments (?100-bp), as well as technologies that can repair formalin adducts, will go a long way toward bringing these clinical specimens to the forefront of disease biomarker discovery and characterization.
About the Author
Steve Michalik works at Sigma-Aldrich as Senior Scientist specializing in whole genome amplification and archived tissue analysis. He has developed several products for purification, amplification, and analysis of nucleic acids from archived samples.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 3, March, 2008, pp. 24-30.
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Filed Under: Drug Discovery
