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Cat. No. / ID: 151023
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✓ Knowledgeable and professional Product & Technical Support
✓ Fast and reliable (re)-ordering
The REPLI-g Mitochondrial DNA Kit enables selective amplification of mitochondrial DNA from total DNA samples, without the need for prior mitochondrial DNA isolation. This kit provides DNA polymerase, buffers, and reagents for specific and uniform whole genome amplification from small samples of human and non-human mitochondrial DNA in total DNA samples. For amplification of non-human mtDNA, the REPLI-g Human mt Primer Mix needs to be substituted with an appropriate primer mix suitable for that species (not provided with the kit). The REPLI-g Mitochondrial DNA Kit is based on the multiple displacement amplification technology (MDA), that enriches for mitochondrial DNA with minimal contamination from nuclear DNA, thus avoiding the need for time-consuming isolation of mitochondrial DNA and increasing the sensitivity of downstream assays.
One limitation to the use of mitochondrial DNA has been the need to isolate it from nuclear DNA, particularly in cases where the sensitivity of mitochondrial DNA marker assays needs to be increased. The isolation procedure involves many time-consuming steps and leads to substantial losses of mitochondrial DNA. The REPLI-g Mitochondrial DNA Kit overcomes this limitation by enriching for mitochondrial DNA sequences in total DNA samples.
REPLI-g Mitochondrial DNA technology provides fast and highly uniform DNA amplification across the entire mitochondrial genome. The method is based on multiple displacement amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase. Due to its high processivity and strand displacement activity, REPLI-g DNA Polymerase is capable of amplifying up to 100 kb without dissociating from the DNA template and minimizes unequal sequence and locus representation compared with PCR-based amplification methods.
REPLI-g amplified mitochondrial DNA can be used in a variety of downstream applications, including:
|Amplification||Whole genomic DNA|
|Samples per run (throughput)||Mid|
|Maximum input volume||10 µl template DNA|
|Minimal pipetting volume needed||1 µl|
|Reaction volume||50 µl|
|Reaction time||~8 hours (overnight)|
|Applications||Genotyping, sequencing, RFLP|
|Starting amount of DNA||~10 ng purified total DNA|
|Starting material||Genomic human DNA|
|Technology||Multiple Displacement Amplification (MDA)|
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 .
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.
Intact mitochondrial DNA is necessary for successful amplification with the REPLI-g Mitochondrial DNA Kit. Most DNA extraction methods are optimized for the isolation of linear nuclear DNA rather than for mitochondrial DNA. Furthermore, any harsh conditions leading to fragmentation of double stranded DNA must be avoided. We recommend the use of QIAamp DNA Kits, which are available in various formats and optimized for different sample materials.
Low yields with REPLI-g Kits for whole genome amplification (WGA) can result from a number of factors:
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!
It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.
Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA as anticoagulant to prevent the release of genomic DNA into the plasma fraction.
Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.
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.
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.
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.
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.
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.
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).