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Ampli1™ WGA Kit FAQs

How much input material can be amplified using Ampli1™ WGA Kits?

The Ampli1™ WGA protocol is optimized for amplification of the DNA content of one single cell; however, the kit works equally well to amplify DNA from multiple cells.


Can DNA from fixed or preserved cells be amplified with Ampli1™ WGA Kits?

Silicon Biosystems has validated that preservatives, such as that used in Veridex CellSave tubes for the CellSearch® System, or fixatives, such as 1–2% PFA, have little effect on the efficiency of amplification with Ampli1™ WGA Kits. Stronger fixatives may lead to lower yields.

For questions regarding compatibility of Ampli1™ WGA Kits with other fixatives, please contact us.


How much material is generated from a single cell amplified with the Ampli1™ WGA Kits?

Ampli1™ WGA amplification generally produces about 2 micrograms dsDNA and 5 micrograms of ssDNA. When required by the application, the ssDNA product can be converted to dsDNA, producing a total of up to about 12 micrograms dsDNA.


What is the size of the DNA fragments that are generated by Ampli1™ WGA Kits?

Amplified DNA fragments range in size from 0.1–2kb.


How does Ampli1™ WGA work?

Ampli1™ workflow is in 4 steps: 

  1. Genomic DNA digestion with a frequent cutter restriction enzyme, to generate exactly 19,046,047 fragments, all ending with the same sticky end on both sides
  2. Ligation of a single adaptor on both  overhangs of each fragment
  3. Fill-in reaction to complement the sequence of the adaptor
  4. Amplification of the entire genome library with one single high specific PCR primer corresponding to the adaptor

Is the genomic amplification exponential?

Yes. The Ampli1™ WGA Kit uses a standard PCR amplification mediated by a single highly specific primer to ensure minimum bias across the genome and exact replication of both alleles at each PCR cycle.


How is the Ampli1™ method different from other WGA technologies?

Unlike other WGA technologies based on PCR, the Ampli1™ method does not rely on random fragmentation of genomic DNA. The restriction enzyme-mediated fragmentation used in the Ampli1™ protocol results in low drop-out rate and balanced amplification, since the two alleles of any given locus will generate amplicons of identical size and GC content.

Unlike other WGA technologies based on random priming, the Ampli1™ method uses a single highly specific primer, thus avoiding the complexity of multiple binding sites and the introduction of errors due to mispriming.

Unlike other WGA technologies based on strand displacement amplification, the Ampli1™ method is very resilient to fixation, as the impact of DNA cross-linking or damage at a given locus is limited to the specific gDNA Ampli1™ fragment containing that locus.


What downstream analyses can be performed with Ampli1™ WGA-amplified DNA?

Amplified DNA may be used for downstream analyses, of SNPs and CNV detection with a variety of techniques including: 

  • Sanger Sequencing
  • Next generation sequencing
  • Real Time PCR
  • Array CGH 

If locus specific PCR has to be performed on WGA product to generate templates for DNA sequencing or other analyses, verify the absence of a restriction site between the two PCR primers on genomic DNA sequence. In case a restriction site is present between the two primers one of the two primers must be repositioned on the other side of the cutting point to avoid PCR failure.


Can I use the Ampli1™ method on extreme dilutions of DNA?

Yes, the Ampli1™ kit can be used to amplify extreme dilutions of purified DNA; however, SNP calling will be compromised by the stochastic unbalance of alleles in the sample. Only CNV analysis can be reliably conducted on such samples.


 The Ampli1™ WGA Kit is for Research Use Only. Not for use in diagnostic procedures.

Please contact us if you have questions about the compatibility of Ampli1™ WGA-amplified DNA with your downstream application.

 

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