REPLI-g FFPE Kit

For direct whole genome amplification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue

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REPLI-g FFPE Kit (25)

Cat. No. / ID: 150243

DNA Polymerase, Buffers, and Reagents for 25 x 50 μl whole genome amplification reactions

Kit

REPLI-g FFPE Kit (25)

REPLI-g FFPE Kit (100)

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The REPLI-g FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

Whole genome amplification directly from paraffin sections
Whole genome amplification of purified DNA from FFPE tissue
Scalable and standardized DNA yields: up to 40 µg per tissue section
Fast and easy protocol for rapid results

Product Details

A lack of sufficient quantities of genomic DNA for genomic analysis can be overcome by global amplification of all DNA within a sample (whole genome amplification). The REPLI-g FFPE Kit consists of DNA Polymerase, novel buffers, and reagents that enable efficient whole genome amplification from formalin-fixed and paraffin embedded (FFPE) tissue without the requirement for prior DNA purification. After lysis of the tissue section, the DNA is processed so that fragmented DNA is ligated. The long DNA strands created are amplified using proven REPLI-g technology.

Performance

For the two protocols with different amplification times, typical yields per 50 µl reaction with standard quality templates are up to 10 µg after 2-hour amplification (standard reaction) and up to 40 µg after 8-hour amplification (high-yield reaction).

Principle

The availability of sufficient quantities of genomic DNA for genomic analysis is often lacking. Whole genome amplification (WGA) overcomes this limitation by global amplification of all DNA within a sample, providing sufficient quantities to perform all analyses on the same DNA sample.

Genotyping of formalin-fixed, paraffin-embedded (FFPE) tissue samples enables morphological tissue changes to be directly linked to specific genome mutations. However, formalin fixation causes irreversible damage to the DNA, resulting in fragmented DNA that is cross-linked to other biomolecules within the sample. In addition, the limited amount of DNA that can be extracted from FFPE tissue samples is only sufficient for a few analyses.

The REPLI-g FFPE Kit overcomes these problems through a procedure involving a novel DNA processing reaction that prepares and ligates fragmented DNA (see figure " REPLI-g FFPE principle"). Whole genome amplification of this randomly ligated DNA is then performed using proven REPLI-g technology. REPLI-g products combine multiple displacement amplification (MDA) with a uniquely processive DNA polymerase. Due to the high processivity and strand displacement activity, REPLI-g DNA Polymerase minimizes unequal sequence and locus representation. This provides much more reliable results when compared to PCR-based WGA methods. The novel DNA processing reaction allows the advantages of MDA to be extended to highly degraded DNA samples derived from FFPE tissue. Precious sample material can be amplified whilst maintaining locus representation, enabling unlimited downstream analyses to be performed.

See figures

REPLI-g FFPE principle.

Procedure

The REPLI-g FFPE protocol allows the amplification of purified DNA from FFPE tissue samples or DNA direct from FFPE tissue samples without prior DNA purification. After lysis of the tissue section, the DNA is processed using novel buffers and enzymes that ligate fragmented DNA (see flowchart " REPLI-g FFPE procedure"and figure " High-molecular-weight ligated DNA"). The long DNA strands created by the ligation reaction are amplified using proven REPLI-g technology. Once amplified, the DNA is suitable for immediate use in most downstream genotyping assays without further purification.

DNA amplified using the REPLI-g FFPE procedure is highly suited for use in real-time PCR (e.g., using QuantiFast Kits) and end-point PCR (e.g., using the QIAGEN Fast Cycling PCR Kit), with a PCR amplicon preferably smaller than the average fragment size of the starting template. Further applications are microsatellite analysis (see figure " Reliable microsatellite analysis") and SNP genotyping (REPLI-g FFPE amplified DNA may not be suited for genotyping methods that require restriction digestion for DNA labeling).

See figures

REPLI-g FFPE procedure.

High-molecular-weight ligated DNA.

Reliable microsatellite analysis.

Applications

DNA amplified using the REPLI-g FFPE procedure is highly suited for use in:

Real-time PCR
End-point PCR
SNP genotyping
Microsatellite analysis

Supporting data and figures

REPLI-g FFPE procedure.

Formalin fixation and paraffin embedding cause DNA fragmentation. During the REPLI-g FFPE procedure, these DNA fragments are randomly ligated and then amplified using multiple displacement amplification. As indicated, DNA fragments (containing different loci [L1–L4]) are not assembled in the original order. However, the different loci are equally amplified and can be detected in downstream genotyping applications.

Specifications

Features	Specifications
Yield	10 µg (2h reaction)-40 µg (8h reaction)
Quality assessment	No
Starting material	FFPE tissue sections or purified genomic DNA
Technology	MDA
Maximum input volume	1 tissue section (10-40 µm thickness)
Reaction time	2-8h (2h: standard; 8h: high-yield reaction)
Reaction volume	50 µl
Starting amount of DNA	100-300 ng*
Applications	Genotyping, PCR, Real-time PCR
Samples per run (throughput)	medium
Amplification	Whole genomic DNA

Resources

Sample to Insight solutions for successful molecular analysis

High-quality, nucleic acid purification for successful PCR and NGS experiments.

Second edition — innovative tools

FFPE サンプルの分子解析における重要なファクター

High-quality, nucleic acid purification for successful PCR and NGS experiments.

Critical factors for molecular analysis of FFPE samples

For direct whole genome amplification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue

Publications

Genome-wide DNA methylation analysis of archival formalin-fixed paraffin-embedded tissue using the Illumina Infinium HumanMethylation27 BeadChip.

Thirlwell C; Eymard M; Feber A; Teschendorff A; Pearce K; Lechner M; Widschwendter M; Beck S;

Methods; 2010; 52 (3):248-54 2010 Apr 29 PMID:20434562

Whole genome amplification for array comparative genomic hybridization using DNA extracted from formalin-fixed, paraffin-embedded histological sections.

Huang J; Pang J; Watanabe T; Ng HK; Ohgaki H;

J Mol Diagn; 2009; 11 (2):109-16 2009 Feb 5 PMID:19197000

FAQ

The REPLI-g Whole Genome Amplification (WGA) method is a rapid and reliable method of generating unlimited DNA from a few cells or a few nanograms of genomic DNA. This technology amplifies the genome with comprehensive loci coverage and minimal bias between any loci, yielding 12+ kb fragments in a simple, scalable reaction.

FAQ ID -654

The REPLI-g amplified products can be used directly for downstream analysis such as PCR, PCR-based applications, restriction enzyme digestion, cycle sequencing, and more, after appropriate dilution to adjust to work concentrations.

However, to determine DNA concentration by absorbance, the MDA product should be run through a spin column to eliminate the random hexamers, as they will contribute to the absorbance reading and give an artificially high concentration. For this reason, we recommend determining DNA concentration by PicoGreen analysis, which preferentially binds double-stranded DNA. As a result, single-stranded random hexamers will not contribute to the apparent DNA concentration in the quantitation assay. When using this method, the concentration of the MDA product can be determined directly, without any purification.

A Protocol for the use of PicoGreen to quantitate REPLI-g WGA product can be found in the REPLI-g Mini/Midi Handbook. Please follow this link .

FAQ ID -713

These regions are not amplified because only a subset of the random primers in the REPLI-g amplification mix can prime within these extensively repeated regions. Despite the high processivity of the Phi29 DNA Polymerase, centromeres and telomeres are at a competitive disadvantage during amplification, and drop out. In experiments we have done, single copy sequences within approximately 5000 bases of a poorly amplified region can be affected during amplification. If your gene is further than 5000 bases apart from a centromere or telomere, it should be amplified just fine.

FAQ ID -702

Yes. Chromosomes are equally amplified. We and our customers use amplified DNA for SNP genotyping on a regular basis, using Illumina, TaqMan®, Sequenom, PCR, gel-based sequencing, and other techniques. Tzvetkov et al. (2005) used Affymetrix’ GeneChip Human Mapping 10K Arrays to investigate the accuracy and allele amplification bias in DNA samples subjected to MD-WGA with REPLI-g. They observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. Genomic DNA for this study was extracted from blood samples of four unrelated donors using the QIAamp DNA Blood Kit. High-throughput microarray genotyping of 11 555 different SNPs was performed using GeneChip Human Mapping 10K Arrays version Xba131 (Affymetrix).

For additional references, please visit our continuously expanding Citation Data Base online.

See trademarks.

FAQ ID -700

Low yields with REPLI-g Kits for whole genome amplification (WGA) can result from a number of factors:

inhibitors in the template DNA, e.g., phenol and SDS, EDTA > 1 mM
low-quality, fragmented input DNA, e.g., degraded DNA from old samples, FFPE samples
improper reaction conditions, e.g., wrong volumes pipetted, insufficient reagent mixing, denaturation and neutralization steps omitted, or carried out incorrectly
Thermocycler incorrectly programmed, e.g., incubation time set incorrectly, reaction temperature too high (heated lid not adjusted to 70°C)

Note! When using a thermocycler model that does not allow adjusting the temperature of the heated lid, REPLI-g incubation temperature has to be reduced to 25-28°C to ensure optimal reaction conditions and amplification efficiency!

FAQ ID -2148

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.

FAQ ID -1756

We do not sell any REPLI-g reaction components separately.

FAQ ID -708

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.

FAQ ID -673

These are DNA primers of random sequence, six nucleotides long, with two thiophosphate linkages at the 3' terminus to prevent digestion of the oligos by the 3' to 5' exonuclease activity of Phi29 Polymerase.

FAQ ID -710

The reaction works at 30^oC and will not work efficiently at higher temperatures. This is because the Phi29 DNA polymerase is not a thermostable enzyme and the random hexamer primers bind less efficiently as temperature is increased.

FAQ ID -656

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

FAQ ID -694

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.

FAQ ID -701

The REPLI-g kit is designed for whole genome amplification using random hexamers. Addition of specific primers instead of random hexamers may introduce amplification bias, or the preferential amplification of specific DNA fragments at the expense of others. Currently, specific primers alone cannot be used to amplify a specific region of the genome with REPLI-g.

FAQ ID -712

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.

FAQ ID -1758

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.

FAQ ID -1690

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.

FAQ ID -690

After the REPLI-g reactions are completed, 1 ug of the WGA reaction product can be analyzed by electrophoresis through a 1.0% agarose gel in TBE buffer (90 mM Tris-borate, pH 8.0, 2 mM EDTA), stained with GelStar, ethidium bromide, or SYBR Green, and imaged with a UV-box or a Phosphor-Imager. The majority of product should be above 10 Kb in length, and generate a trail of smear by electrophoresis down to about 2 Kb.

FAQ ID -695

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.

FAQ ID -675

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).

FAQ ID -1760

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.

FAQ ID -1753

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.

FAQ ID -1754

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.

FAQ ID -707

We have been conducting an ongoing stability study for more than a year without observing breakdown of the amplified product. There is nothing in the amplification product that indicates that it would not be stable for a number of years.

FAQ ID -693

The enzyme used in the REPLI-g reaction is Phi29 DNA Polymerase.

FAQ ID -704

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.

FAQ ID -1755

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.

FAQ ID -682

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.

FAQ ID -665