REPLI-g Mitochondrial DNA Kit

For highly uniform whole genome amplification from human and non-human mitochondrial DNA

Features

  • Sensitive amplification from varying sample types
  • Suitable for amplification of human and non-human mtDNA
  • Overcomes the need for time-consuming isolation of mtDNA
  • Informative results from samples with degraded nuclear DNA
  • DNA amplification from blood cards and hair
REPLI-g Mitochondrial DNA Kit (25)

Cat. No. / ID: 151023

DNA Polymerase, Buffers, and Reagents for 25 x 50 μl mitochondrial DNA specific whole genome amplification reactions
€244.00
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The REPLI-g Mitochondrial DNA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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.

Performance

Total DNA preparations typically comprise approximately 0.1% of mitochondrial DNA (e.g., 0.1 ng of total human DNA contains 0.1 pg of mitochondrial DNA). The REPLI-g Mitochondrial DNA Kit provides specific amplification of the whole human mitochondrial genome, and yields approximately 4 µg of amplified mitochondrial DNA per reaction. This corresponds to an enrichment of mitochondrial DNA of up to 40 million-fold, depending on the starting amount (see figure " Enrichment of mitochondrial DNA"). Residual amounts of amplified genomic DNA are minimized and do not interfere with mitochondrial-specific downstream methods such as qPCR or direct Sanger sequencing.
See figures

Principle

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.

Procedure

Amplification of mitochondrial DNA using the REPLI-g Mitochondrial DNA Kit involves just two basic steps (see flowchart " Purified mitochondrial DNA procedure"). First, the total human DNA sample is denatured by incubation in REPLI-g mt Reaction Buffer and REPLI-g Human mt Primer Mix for 5 minutes at 75°C and then cooled to stop the denaturation. However, to denature DNA sample from non-human species, the REPLI-g Human mt Primer Mix should be substituted with the appropriate primer mix for that species (not included in the kit). Next, REPLI-g DNA Polymerase is added and the isothermal amplification reaction proceeds for 8 hours at 33°C.
See figures

Applications

REPLI-g amplified mitochondrial DNA can be used in a variety of downstream applications, including:

  • Genotyping (e.g., SNP, deletions, insertions)
  • Endpoint-PCR, quantitative real-time PCR 
  • Sequence analysis

Supporting data and figures

Specifications

FeaturesSpecifications
amplificationWhole genomic DNA
samplesperrunthroughputMid
denaturationstepHeat
maximuminputvolume10 µl template DNA
minimalpipettingvolumeneeded1 µl
reactionvolume50 µl
reactiontime~8 hours (overnight)
qualityassessmentNo
applicationsGenotyping, sequencing, RFLP
startingamountofdna~10 ng purified total DNA
startingmaterialGenomic human DNA
technologyMultiple Displacement Amplification (MDA)
yield~4 µg

Publications

Mitochondrial DNA mutation in normal margins and tumors of recurrent head and neck squamous cell carcinoma patients.
Dasgupta S; Koch R; Westra WH; Califano JA; Ha PK; Sidransky D; Koch WM;
Cancer Prev Res (Phila); 2010; 3 (9):1205-11 2010 Jul 26 PMID:20660573

FAQ

What is REPLI-g whole genome amplification?
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
Will the random hexamers in the REPLI-g reaction interfere with downstream analysis?

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
Are Centromeres and Telomeres amplified using REPLI-g WGA?
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
Can I use REPLI-g for SNP Genotyping?

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
Can I use any type of DNA extraction method to purify template DNA for amplification using the REPLI-g Mitochondrial DNA Kit?

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.

 

FAQ ID -1321
What are possible reasons for reduced DNA yields with REPLI-g Kits?

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
Can I purchase Phi29 DNA polymerase only?
We do not sell any REPLI-g reaction components separately.
FAQ ID -708
Does REPLI-g technology for whole genome amplification work with paraffin embedded samples?

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
What are exo-resistant random hexamers used in the REPLI-g reaction?
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
Will REPLI-g work at high temperatures?
The reaction works at 30oC 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
How can I quantify the amount of REPLI-g DNA I have amplified?
  • 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
Why is there DNA in the no-template control reaction when using the standard REPLI-g procedure, but not when using the UltraFast procedure?

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.

 

FAQ ID -1327
Has anyone verified whole genome amplification accuracy with Sequencing?

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
Can I use my own primers for REPLI-g WGA to amplify a specific chromosomal region?
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
Can the REPLI-g Mitochondrial DNA Kit be used for species other than humans?

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.

FAQ ID - 3584
Where can I find background information and literature on Whole Genome Amplification with REPLI-g Kits?

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
What is the length of the MDA whole genome amplification product?

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
How can I determine the quality of my REPLI-g amplified products?
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
Why do I get amplification in a negative control DNA tube using the REPLI-g Kit for WGA?

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
How much REPLI-g amplified mitochondrial DNA should be used in a subsequent PCR reaction?

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.

 

FAQ ID -1320
Do mutations in the target mitochondrial DNA prevent primer binding, resulting in no DNA amplification with the REPLI-g Mitochondrial DNA Kit?

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.

 

FAQ ID -1323
Any data on the fidelity of the REPLI-g MDA technique?

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
What is the stability of the REPLI-g MDA product?
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
What is the enzyme used in the REPLI-g reaction?
The enzyme used in the REPLI-g reaction is Phi29 DNA Polymerase.
FAQ ID -704
Can I amplify mitochondrial DNA directly from whole blood or cells using the REPLI-g Mitochondrial DNA Kit?

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.

 

FAQ ID -1319
Can I do whole genome amplification from mitochondrial DNA using REPLI-g?
Yes, we offer the REPLI-g Mitochondrial DNA Kit for this application.
FAQ ID -668
Is 2 kb the minimal gDNA fragment size for REPLI-g Whole Genome Amplification?

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
What are the differences between MDA and DOP/PEP methods of Whole Genome Amplification?

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
Do you have a protocol for cleanup of REPLI-g amplified DNA?

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

 

 

 

FAQ ID -1545