REPLI-g Mitochondrial DNA Kit

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!

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

In no-template (negative) control reactions, primer-dimers can form. The highly processive Phi29 DNA Polymerase will extend these primer-dimers leading to unspecific amplification products during the long incubation time with the standard REPLI-g procedure. Amplification in no-template controls takes place much more slowly than the amplification of template DNA. Using the REPLI-g UltraFast Mini Kit, the incubation time is too short to allow the extension of primer-dimers.

In any case, non-specific amplification products will not compromise results in downstream genetic analysis and do not appear if template DNA is present.

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.

Yes, the REPLI-g Mitochondrial DNA Kit can be used for amplifying mitochondrial DNA from humans as well as other species. When the kit is used to amplify mitochondrial DNA from other species than humans, users can substitute the REPLI-g Human mt Primer Mix with an appropriate primer mix of their choice.

The efficiency and specificity of amplification is, in part, dictated by the sequence of the primers. The success of the amplification reaction using the REPLI-g Mitochondrial DNA Kit is greatly enhanced when the primers used for amplification are designed according to the following rules:

1. Select 10 to 20 different primers from the mitochondrial genome of interest.
2. Ensure that half of the primers hybridize to one strand, while the second half hybridizes to the complementary strand.
3. Select an almost uniform distance between primer hybridization sites within the mitochondrial genome.
4. Use primers that are 8 to 14 nt long.
5. Use a phosporothioate (PTO) backbone at the 3’ end of the primers. We recommend including phosphorothioate modifications between the last 3 bases of the 3’ end of the primers: 5’-N-N-----------------N*N*N-3’.
6. Ensure that the concentration of primers is optimized to be in the range of 1 to 10 µM.

Please be aware that for other REPLI-g products the primers are included in the master mix and cannot easily be exchanged.

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.

The REPLI-g Mitochondrial DNA Kit amplifies mitochondrial DNA approximately 40 million-fold. The small size of the mitochondrial genome leads to very high copy numbers per microliter of amplified DNA. Therefore, we recommend dilution of the amplified DNA 1:1000 with nuclease-free water. Just 1 µl of the diluted sample should be used in a single PCR reaction.

No, there is no risk that mitochondrial DNA will not be amplified due to a change in the primer binding site (i.e., caused by a deletion). This is because several primers are used in the amplification reaction with the REPLI-g Mitochondrial DNA Kit.

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.

No, currently you need to purify total DNA first before using the REPLI-g Mitochondrial DNA Kit. For purification of total DNA, we recommend QIAamp DNA Kits, which are available in various formats and optimized for different sample materials.

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.

Yes, please follow the Supplementary Protocol 'Purification of REPLI-g amplified DNA using the QIAamp DNA Mini Kit' (RG14).