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Single Cell whole genome amplification on magnetic microbeads
Hello forum members,

We are looking to bind single cell genomic DNA to microbeads and perform a whole genome amplification on the beads. We want to perform a non PCR ampificiation.

The idea is to bind the DNA to the microbeads using the streptadivin - biotin system. We need the DNA bound to microbeads while leaving the DNA available for amplification, thus we want to bind the fosfate backbone with DNA binding proteins.

I have a few questions:

1) Do you think it is feasable to perform whole genome amplfication on microbeads?

2) Can annyone suggest a DNA binding protein that we could biotinylate to produce a probe that leaves the bound DNA available to amplification?

3) Do you have a different suggestion as to how we could bind DNA to microbeads, and subsequently perform a DNA amplification?

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Dear Mandokir,
Thanks for your query.
The DNA amplification using microbeads can be feasible but at the same time it also depends on the requirements of your project. Kindly find below comparision of standard methods of DNA amplification and thier cost.

454 Sequencing -Cost per run-$ 8,438 USD (Approach-Emulsion PCR )
Illumina - Cost per run - $ 11,750 USD (Approach-Bridge Amplification)
SOLiD -Cost per run - $ 17,477 USD. (Approach-Emulsion PCR )

You can also compare your method cost with these standards.

Further to immobilize/ bind DNA to microbeads, Polish (make blunt at each end ) smaller fragments approx -300-800 base pairs. Ligate Short adaptors onto the ends of the fragments. These adaptors provide priming sequences for both amplification and sequencing of the sample-library fragments. One adaptor (Adaptor B) contains a 5'-biotin tag for immobilization of the DNA library onto streptavidin-coated beads. After nick repair, the non-biotinylated strand is released and used as a single-stranded template DNA (sstDNA) library. The sstDNA library is assessed for its quality and the optimal amount (DNA copies per bead) eitehr by titration.

Thus sstDNA library is immobilized onto beads.
The beads containing a library fragment carry a single sstDNA molecule. The bead-bound library is emulsified with the amplification reagents in a water-in-oil mixture. Each bead can then be captured within its own microreactor. The available fragment can be amplified using enzyme bacteriophage Φ29 DNA polymerase that can produce DNA product of 7kb to 10kb long. Its high fidelity and 3’–5' proofreading activity reduces the amplification error rate also to 1 in 106−107 bases compared to conventional Taq polymerase

1] The reaction can be carried out at a moderate isothermal condition of 30°C and therefore exempts the needs of the thermocycler. It has been actively used in cell-free cloning methods also, which is the enzymatic method of amplifying DNA in vitro without cell culturing and DNA extraction. The large fragment of Bst DNA polymerase is also used in MDA, but Ф29 enzyme is generally preferred due to its sufficient product yield and proofreading activity.

Thus this results in bead-immobilized, clonally amplified DNA fragments.

To answer your third question, i would suggest you to use
“LAMP" technique which stands for Loop-mediated Isothermal Amplification is a simple, rapid, specific and cost-effective nucleic acid amplification method and a good alternative for PCR amplification.
This is solely developed by Eiken Chemical Co., Ltd.

It is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature using strand displacement reaction.

Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature (about 65°C).

It provides high amplification efficiency, with DNA being amplified 109-1010 times in 15-60 minutes.

Because of its high specificity, the presence of amplified product can indicate the presence of target gene.
Some of the special characterists of this techniques are :-

• There is no need for a step to denature double stranded into a single stranded form.
• The whole amplification reaction takes place continuously under isothermal conditions.
• The amplification efficiency is extremely high.
• By designing 4 primers to recognize 6 distinct regions, the LAMP method is able to specifically amplify the target gene.
• The total cost can be reduced, as LAMP does not require special reagents or sophisticated equipments.
• The amplified products have a structure consisting of alternately inverted repeats of the target sequence on the same strand.
• Amplification can be done with RNA templates following the same procedure as with DNA templates, simply through the addition of reverse transcriptase.

When the target gene (DNA template as example) and the reagents are incubated at a constant temperature between 60-65°C, the following reaction steps proceed:

As double stranded DNA is in the condition of dynamic equilibrium at the temperature around 65°C, one of the LAMP primers can anneal to the complimentary sequence of double stranded target DNA, then initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and releasing a single stranded DNA. With the LAMP method, unlike with PCR, there is no need for heat denaturation of the double stranded DNA into a single strand. The following amplification mechanism explains from when the FIP anneals to such released single stranded template DNA.

Through the activity of DNA polymerase with strand displacement activity, a DNA strand complementary to the template DNA is synthesized, starting from the 3' end of the F2 region of the FIP.

The F3 Primer anneals to the F3c region, outside of FIP, on the target DNA and initiates strand displacement DNA synthesis, releasing the FIP-linked complementary strand.

A double strand is formed from the DNA strand synthesized from the F3 Primer and the template DNA strand.

The FIP-linked complementary strand is released as a single strand because of the displacement by the DNA strand synthesized from the F3 Primer. Then, this released single strand forms a stem-loop structure at the 5' end because of the complementary F1c and F1 regions.

This single strand DNA in Step (5) serves as a template for BIP-initiated DNA synthesis and subsequent B3-primed strand displacement DNA synthesis. The BIP anneals to the DNA strand produced in Step (5). Starting from the 3' end of the BIP, synthesis of complementary DNA takes place. Through this process, the DNA reverts from a loop structure into a linear structure. The B3 Primer anneals to the outside of the BIP and then, through the activity of the DNA polymerase and starting at the 3' end, the DNA synthesized from the BIP is displaced and released as a single strand before DNA synthesis from the B3 Primer.

Double stranded DNA is produced through the processes described in Step (6).

The BIP-linked complementary strand displaced in Step (6) forms a structure with stem-loops at each end, which looks like a dumbbell structure. This structure serves as the starting structure for the amplification cycle in the LAMP method (LAMP cycling). The above process can be understood as producing the starting structure for LAMP cycling.

Thank you once again for putting your questions in this forum.
I hope this will help you & Best of luck for your project !

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Thank you for your answer,

However i belive that conventional DNA amplification methods are unsuitable for our research.

The single cells are trapped inside a cilinder shaped container with a vollume of 1.5nl. We cannot remove the cells, we can only add reagents. However when we attempt to lyse the cells DNA will be lost if it cannot be bound to something, so we add DNA binding beads.

Next we want to perform a DNA amplification, the problem is that we cannot remove the beads efficiently either. Thus we want to perform the DNA amplification inside the 1.5nl cilinders. We cannot perform a PCR due to isuues with the carrier material. Thus we need to perform a non PCR amplification.

So we are bound to the folowing restrictions:

- We cannot remove the genomic DNA bound to the beads
- We cannot perform PCR
- We cannot perform anny emulsion techniques
- The amplification has to be performed in a 1.5nl vollume
- We need to amplify the whole genome

Thus: can we amplify whole genomic DNA bound to beads in anny way with the above restrictions aplied?

What would happen if we just add amplification reagents to the DNA bound to the beads? Would the DNA be amplified? Would the formed DNA also stick to the beads?

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Wonderful answer. Thanks for sharing everything in brief. Your step by step procedure is really helpful.
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(04-03-2013, 09:29 PM)brianrey Wrote: Wonderful answer. Thanks for sharing everything in brief. Your step by step procedure is really helpful.
genomic dna

Dear Brianrey, thanks for appreciating my answer.

Yes, these steps really give clear idea on what happens after target gene and the reagent are incubated, at a constant temperature which is between 60-65°C.

Please share, if any other query on this topic. We can always discuss !

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