REPLI-g FFPE Kit

A single tissue section between 10-40 µm thickness is recommended for use per reaction with the REPLI-g FFPE Kit. For additional details, please refer to the protocol in the REPLI-g FFPE Handbook.

Standard REPLI-g Kits, such as the REPLI-g Mini and Midi, UltraFast Mini-, Screening-, and Mitochondrial DNA Kits are not recommended for the amplification of gDNA extracted from paraffin embedded tissues. DNA recovered from paraffin embedded samples is typically strongly fragmented due to the fixation process, resulting in fragments only a few hundred base pairs long. Phi29 DNA polymerase however works most efficiently on DNA longer than 2 kb in length (ideally, at least a few fragments >10 kb should be present in the gDNA sample). If WGA product from strongly fragmented starting samples is utilized in genotyping assays, significant allele drop out and mis-genotyping can occur.

However, our new REPLI-g FFPE Kit overcomes these limitations by pre-processing of DNA directly derived from paraffin embedded tissue samples. The pre-processing reaction ligates fragments to generate suitable templates for subsequent amplification with REPLI-g Midi DNA Polymerase.

Further information can be found in the WGA Tutorial on our WGA Resource page.

• Since REPLI-g amplification products contain single-stranded DNA as well as residual primers, it is important to utilize a DNA quantification method that is specific for double-stranded DNA. PicoGreen is a fluorescence-based nucleic acid quantitation method that is specific for double-stranded DNA and may be used to quantify the double-stranded REPLI-g products. For best results, the sample should be diluted with 2 volumes of water and thoroughly mixed prior to addition of PicoGreen.
• before taking an OD reading on a spectrophotometer the reaction product must be purified as it contains unused primers and dNTPs

Paez et al. 2004 have shown in direct sequencing experiments sampling 500 000 bp, that the estimated error rate (9.5 x 10^-6) was the same in WGA generated samples as in paired unamplified samples.

When using PicoGreen for quantification of REPLI-g FFPE amplifiied DNA, perform the assay after step 15 of the REPLI-g FFPE protocol, prior to the heating step at 95°C.

PicoGreen detects double-stranded DNA with high sensitivity. If the DNA was quantified after denaturation at 95°C using PicoGreen, multiply the yield by a factor of 2 to compensate for the use of single-stranded DNA.

Please visit our Whole Genome Amplification Resource Page for access to comprehensive information on WGA using REPLI-g Kits and REPLI-g Services.

Our WGA tutorial provides further information about Whole Genome Amplification and discusses the various techniques that are used. Additional detailed information is provided about REPLI-g Multiple Displacement Amplification technology (MDA), and recommendations are given on how to achieve the best results.

MDA-amplified product has an average length of 10-12 kb, enabling Southern Blots, RFLP, and other downstream analyses that require large DNA fragments.

Note that the ligation procedure employed in the REPLI-g FFPE Kit results in the formation of very high-molecular-weight DNA and enables uniform whole genome amplification from formalin-fixed, paraffin-embedded (FFPE) tissue.

Further information on yield and length of amplified DNA can be found in the WGA Tutorial on our Whole Genome Amplification Resource Page.

Phi29 DNA Polymerase has an extremely high processivity and will extend primer-dimers that may be present in the reaction, leading to unspecific amplification products. Furthermore, the REPLI-g reaction is highly sensitive to any traces of DNA. Even minute quantities of contaminating DNA from other sources are eventually amplified over the long duration of the reaction (6-16h). However, these non-specific products will not generate specific results in downstream genetic analysis.

Formalin-fixed, ethanol-preserved samples that are not embedded in paraffin can be processed with the REPLI-g FFPE Kit by leaving out step 4 and 5 of the REPLI-g FFPE protocol. A mix containing FFPE Lysis Solution* and 2 µl Proteinase K can be added to the tissue section at step 3, after the section has been removed from its preservative solution. Proceed with the protocol at step 7: Incubate for 60 min at 60°C and then for a further 10 min at 95°C.

*Typically, 100 µl FFPE Lysis Solution are used. However, a smaller volume can be used for trimmed tissue sections (see protocol step 3 in the REPLI-g FFPE Handbook for more information).

The novel REPLI-g FFPE technology allows whole genome amplification of fragmented DNA directly from formalin-fixed, paraffin-embedded (FFPE) tissue samples.

The REPLI-g FFPE Kit contains reagents for two different reactions: a processing reaction preparing fragmented DNA from paraffin-embedded tissue for whole genome amplification (WGA) and an amplification reaction. Please refer to the REPLI-g FFPE Handbook for details of the procedure.

No, the REPLI-g FFPE Kit cannot restore DNA derived from FFPE tissue samples in its original order. The sample fixation process results in fragmentation of the DNA, and the original order of fragments is lost.

In the REPLI-g FFPE procedure, fragments are eluted from the tissue section and are ligated randomly during the processing reaction of the protocol. However, this does not effect the detection of nucleic acid sequences, such as polymorhisms, in downstream applications.

Phi29 DNA polymerase is a high fidelity proofreading enzyme and assures a very low replication error rate. It has an error rate of 1 x 10^-6 - 10^-7 nucleotides both in its intrinsic enzymatic activity and during the amplification reaction.

In contrast, Taq DNA polymerase has an intrinsic error rate of approximately 2 x 10^-5 nucleotides, with an accumulation of about one mutation per 900 bases after 20 PCR cycles.

Affymetrix GeneChip Mapping assays require restriction sites for the digestion with enzymes. Due to the procedure of random DNA ligation when using the REPLI-g FFPE Kit, original restriction sites may be lost. Therefore, we do not recommend this kit in combination with the Affymetrix GeneChip procedure.

Affymetrix Targeted Genotyping assays on the other hand use a primer extension protocol and should be compatible with the REPLI-g FFPE procedure as they are not affected by the random ligation process.

Highly degraded samples tend not to be amplified evenly across the genome. In general, an average fragment size of 2 kb in a DNA sample is the lower limit, assuming no portions of the genome are degraded to the point where information will be missing. Often, when the largest fragment in a gDNA sample is 2 kb, other fragments are much smaller and some regions of the genome may have been lost due to degradation. At least a small portion of 10 kb fragments or larger need to be present in the gDNA sample for even amplification of the entire genome.

Further information on working with fragmented DNA can be found in the WGA Tutorial on our Whole Genome Amplification Resource Page.

Note: If you are interested in amplifying DNA from paraffin-embedded samples we recommend to use the REPLI-g FFPE Kit.

DOP (Degenerate Oligonucleotide-primed PCR) and PEP (Primer Extension Preamplification) are PCR-based whole genome amplification (WGA) methods. REPLI-g amplification uses MDA (Multiple Displacement Amplification) which is not a PCR-based method. MDA is scalable with yields adjustable from ug to mg quantities, whereas DOP typically yields 2-3 ug of DNA per reaction. DOP also generates a shorter product which is not suitable for certain downstream applications (e.g. Southern blot and sub-cloning).

DOP and PEP products are different from REPLI-g MDA products for a number of reasons. First, after amplification is complete, PEP products have active thermostable polymerase that will degrade the amplification product over time, because the polymerase cannot be inactivated. Second, PEP reactions consist of PCR amplicons which have the potential to contaminate other reactions as 'runaway amplicons' (e.g., amplicons in the aerosol that may be co-amplified if they accidentally get into other reactions).

REPLI-g product has neither of these issues. The polymerase is heat-inactivated after the REPLI-g reaction is complete, so it cannot digest the amplified product. There are no issues with 'runaway amplicons', because the reaction is performed at constant temperature by a hyper-branching amplification mechanism, amplifying the genome randomly without generating specific amplicons.